Added: Nori Sears - Date: 18.01.2022 23:14 - Views: 47745 - Clicks: 2280
Haemodialysis vascular access dysfunction is currently a huge clinical problem. Although arteriovenous AV fistulae are the preferred mode of dialysis access, they have ificant problems with both early failure to pig av and late fistula failure.
Both are characterized radiologically as a stenosis of the venous segment. Despite the magnitude of the clinical problem, the exact pathogenesis of AV fistula failure remains unclear. The aim of this study was to develop and validate a pig model of AV fistula stenosis and pig av use it to dissect out the mechanisms responsible for this lesion. AV fistulae were created between the femoral artery and vein of Yorkshire Cross pigs.
Animals were sacrificed at 2 days, 7 days, 28 days and 42 days post-surgery. At the time of sacrifice the entire specimen was divided into four regions; the arterial AV-A and venous AV-V portions of the AV anastomosis, the juxta-anastomotic segment JA and the proximal vein PVand assessed for the degree of intima-media thickening and the presence of specific cellular phenotypes.
Haemodynamic parameters were not measured in this set of experiments. ificant luminal stenosis and intima-media thickening were present as early as 28 days and 42 days post-surgery in the pig model. The vast majority of cells within the region of intima-media thickening were myofibroblasts. These studies suggest that early and aggressive intima-media thickening which is made up primarily of myofibroblasts plays an important role in AV fistula stenosis in a pig model of AV fistula placement.
Interventions that target the mechanisms and cellular phenotypes described in this model, may be effective in reducing the very ificant morbidity and economic costs currently associated with AV fistula failure. In an attempt to reduce the very ificant morbidity and economic cost associated with haemodialysis vascular access dysfunction [ 1—5 ], there has recently been a concerted attempt to increase the incidence and prevalence rate of successful arteriovenous AV fistulae in the hemodialysis population [ 6—10 ].
The reasons for non-maturation are not clearly understood [ 1617 ], but are thought to be due to a combination of i an inability of the vein and artery to dilate appropriately in response to increased flow and ii possibly an exaggerated pig av response [ 18 ]. In addition to initial non-maturation, AV fistulae can also develop a later after 3 months stenosis of the venous segment, which is characterized by neointimal hyperplasia [ 19 ]. Thus, although AV fistulae are the preferred form of permanent dialysis access, we are still some distance away from the dream of being able to place a fistula that can support haemodialysis in every patient with end-stage renal disease ESRDregardless of anatomic, demographic or logistical restraints [ 20 ].
The aims of the current study, therefore, were to i develop and validate a pig model of AV fistula stenosis and ii use this model to identify cellular mechanisms involved in AV fistula failure, in order to develop and test out future novel therapies for this condition.
Four-month old, 50 kg Yorkshire Cross domestic swines were purchased from Yeazel and Co. Pigs were anaesthetized and intubated with a combination of xylazine, telazol and atropine. Isoflurane was used for maintenance anaesthesia. Following standard surgical cleansing, a 6-inch skin incision was made just below the inguinal ligament. Fascial and muscle planes were carefully dissected out with attention to haemostasis.
The femoral artery usually about 4 mm and vein usually about 6 mm were identified and dissected out bilaterally. The vein and artery were sized to ensure that there would not be stretching or kinking of the vein following creation of the AV fistula. All visible tributaries of the femoral vein were ligated. The femoral vein was then cut and ligated distally. Due to surgical and other issues the curve of the created AV fistula varied between approximately 30 and 90 degrees in different animals. Vessel loops were then released and a special emphasis was made to obtain excellent haemostasis. Special attention was paid to ensure that there was no kinking or torsion of the completed fistula.
Subcutaneous buprenorphine and fentanyl patches were used for peri- and post-operative analgesia as needed. AV fistula patency was confirmed by auscultation immediately after surgery and then every 3 days. Ten pig av were utilized in this validation study and a total of 16 fistulae were evaluated. Pigs were sacrificed at 2 days four fistulae from two pigs7 days three fistulae from two pigs28 days three fistulae from two pigs and 42 days six fistulae from four pigs time points.
One fistula in the day group and two in the day group was lost due to thrombosis and these were excluded from the study. Neointimal hyperplasia was present in one of the animals which thrombosed after 20d. This animal was pig av included in the analysis due to the confounding effects of thrombosis on neointimal hyperplasia. One pig in the 7-day group had only one fistula placed. At the time of sacrifice the animals were given a lethal injection of sodium pentothal and the entire AV fistula was removed en bloc and placed in formalin.
For the purpose of the current study, we will be focusing only on the AV anastomosis and the venous segment. The entire specimen was then allowed to fix in formalin for 48 h, following which it was coated in paraffin and sectioned as described in Figure 1. For the purpose of this study, the arterial and venous cross sections obtained by sectioning at the a—b level were analysed separately as the arterial AV-A and venous AV-V portions of the AV anastomosis.
Following this, the remaining 2. The first three 4 mm blocks following the AV anastomosis were considered to be the juxta-anastomotic JA segment based on the classification by Beathard et al. While we recognize that such divisions are sometimes arbitrary, we felt that the curved portion of the AV fistula should be within the JA segment. This is different from the generally accepted clinical view of the proximal vein which would extend from the JA region to the central venous system. Three micron thick paraffin-embedded sections were then cut on a pig av and used for the histological, morphometric and immunohistochemical analyses.
Diagrammatic representation of AV fistula. The figure describes the division of the AV fistula into four different regions with capital letters indicating separate tissue blocks with the corresponding lower case letters e—e for example indicating the sites at which the tissue specimen was cut.
Briefly, following deparaffinization and hydration, slides were washed and underwent protease digestion if required for a particular primary antibody. All incubations were performed at room temperature with appropriate washes between each step.
A brown colour indicated a positive stain. Negative controls were performed on each run, by substituting the primary antibody with PBS. In addition, positive control tissue gut, lymph node and spleen was used to document the efficacy of each antibody. We divided the intima-media area by the circumference of the lumen, in order to correct for the degree of dilatation.
Image J software was used for the morphometric analyses see Figure 2.
Percentage LS was calculated by drawing a line around the abluminal border of the intima-media line m, Figure 2followed by a line around the lumen line l in Figure 2. The intima and the media were considered to be a single entity, since in most cases it was impossible to clearly identify an internal elastic lamina. There was also in most cases no real difference at a histological level that would have allowed us to differentiate the neointima from the media.
Morphometric analyses. The morphometric analyses that were performed is described in the figure. An analysis of variance ANOVA analysis was used to identify differences between the same region of the AV fistula at different time points and between different regions at the same time point. Data are presented in Table 1 for differences between the same region at 2 days and 42 days for example, a comparison of LS of PV at 2 days vs 42 days and in Table 2 for differences between different regions of the AV fistula at days only for example, a comparison of LS between the PV and the venous portion of the AV anastomosis at 42 days.
Statistical analysis of differences in percentage luminal stenosis and intima-media area per unit length between the 2 day and 42 days samples in different AV fistula regions. The 2-day value is given first see Figure 3 for standard errors. Statistical analysis of differences in percentage pig av stenosis and intima-media area per unit length between different AV fistula regions at the day time point see Figure 4 for standard errors. Figure pig av describes the progression of intima-media thickening in this model.
At 2 days Figure 3 Athe entire intima-media is only about 3—4 cell layers thick as would be expected in normal vein.
These buds then grow in size and coalesce together pig av result in the very ificant degree of intima-media thickening present at 42 days Figure 3 C and D. Descriptive histology at different time points A — C and within different regions at the same time point C — F : A shows the complete lack of intima-media thickening within the proximal vein PV at 2 days the vessel wall is the region between the two arrows. By 7 days, the PV has developed bud like protuberances Barrows and by 42 days these protuberances have coalesced, to result in a very ificant degree of intima-media thickening double-headed arrows and luminal stenosis.
D documents ificant intima-media thickening double-headed arrow in the juxta-anastomotic JA pig av, albeit in an eccentric pattern minimal thickening at the site of arrowhe. The double arrow in E documents the magnitute of intima-media thickening pig av the vein, while the two arrows do the same for the artery in F.
Note that E and F are different parts of the same section in the plane a—b Figure 1. The asterisks denote the common surgical suture sites. There appeared to be a marked increase in the degree of intima-media thickening at the day time point, from the arterial end of the AV anastomosis to the PV. This is most apparent in the histomorphometric analyses Figure 4 A and Bbut Figure 3 C and F also emphasize this point, with ificant intima-media thickening within the PV 3C and JA segment Figure 3 D as compared to relatively much less thickening within the venous and arterial portions of the AV anastomosis Figure 3 E and F, respectively.
Interestingly, some sections of the PV and JA segment demonstrated a very eccentric pattern of intima-media thickening, with a 40—50 cell layer thickness on one side of the vascular cross section as compared to a complete absence of neointimal hyperplasia on the opposite side of the same cross section Figure 5 E and F.
Histomorphometric analyses. A describes the degree of luminal stenosis within different regions of the AV fistula at different time points. B does the same for intima-media area per unit length. Note that in general with some biological variationthe degree of luminal stenosis and intima-media thickness per unit length increases from day 2 to day 42 and also increases from the arterial end of the AV anastomosis AV-A to the proximal vein PV at the day and day time points.
Scattergrams for luminal stenosis A and intima-media thickening B for individual specimens at different time points. As in all biological systems there is a degree of biological variation between different animals and also between paired AV fistulae in the same animal. This variation appears to be more pronounced for intima-media thickening per unit length as compared to luminal stenosis. Note that i the amount of luminal stenosis and intima-media thickening per unit length, tends to increase in a temporal fashion from 2 days to 42 days and ii at the day and day time points, LS and intima-media thickening tends to be far less within the AV anastomosis as compared to the JA and PV regions.
Interestingly, the venous portion of the AV anastomosis has a ificantly greater degree of luminal stenosis as compared to the arterial portion of the AV anastomosis.
Figure 5 describes representative sections from the proximal vein at 42 days that have been stained with antibodies against SMA, desmin and vimentin. Table 3 describes how these antibodies can be used to identify different cell types.
The vast majority of cells within the region of intima-media thickening appear to be SMA-positive, vimentin-positive and desmin-negative cells, suggesting that they are myofibroblasts [ 22 ]. Of note there are a few desmin-positive cells Figure 3 D within the region of intima-media thickening, which could be contractile smooth muscle cells. Perhaps the most interesting result from our immunohistochemical studies came from the analysis of BrdU staining in cross sections with eccentric intima media thickness.
Thus, Figure 4 F documents an impressive amount of endothelial cell arrow and myofibroblast arrowhead proliferation on the side with intima-media thickening as compared to an almost complete absence of cellular proliferation on the side with minimal pig av thickening asterisk. Cellular phenotyping of cells within the region of intima-media thickening in the pig, AV fistula model. We have described for the first time, the early histological changes that occur within different regions of the venous segment of a pig AV fistula at different time points following the creation of an AV fistula.
We believe that this information about the temporal pattern of intima-media thickening, within different regions of the same AV fistula is an important addition to the already available information on the development of venous stenosis following the creation of a pig AV fistula [ 2223 ]. The from our studies clearly document that i venous stenoses are maximal at specific sites within the venous pig av ii intima-media thickening plays an important role in the pathogenesis of this early venous stenosis and iii the predominant cell type within the areas of venous stenosis is the myofibroblast.
We believe that this work has special relevance in the current clinical climate of an increasing incidence of early AV fistula failure in that it both i identifies potential mechanisms for AV fistula failure and ii provides us with a clinically appropriate model for testing out new therapeutic interventions.Pig av
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Hemodynamic wall shear stress profiles influence the magnitude and pattern of stenosis in a pig AV fistula