The overall goal of the following experiment is to assess the influence of pressure, sheer stress, and external reinforcement on human intimal hyperplasia. This is achieved by simultaneous high pressure perfusion of two segments from the same vein. While one segment is reinforced by an external mesh, the vein segments are then cut into sections for histological and molecular analysis.
These analyses are used to evaluate the effect of an external reinforcement on intimal hyperplasia. The results show that an external reinforcement can reduce intimal hyperplasia that is normally observed after seven days of perfusion. Seno van bypass graft is one of the treatment of choice in patients suffering from peripheral arterial disease.
However, the development of t hyperplasia that leads to graft failure is the major limiting factor in such crafts. This remodeling is due to the adaptation of the van to the arterial environment to develop new therapies. The understanding of molecular mechanisms involved in this phenomenon is essential.
Therefore, we have developed an ex vivo model of perfusion. One of the main advantage of our system is that it allows the study of hemodynamic parameters without any other confounding immune or endocrine factors. An MD PhD student from our laboratory will demonstrate the technique.
Human ous vein samples are obtained from patients undergoing lower limb bypass surgery. The vein is collected and brought to the laboratory under sterile conditions and placed under a laminar flow hood. The minimum size of such veins is nine centimeters long.
The internal diameter is above 2.5 millimeters and the external diameter above four millimeters. The vein is divided into three. Even segments harvested veins are stored in ice cold RPMI 1640 glut max media supplemented with 12.5%FBS rinse one segment in PBS.
It is the control divided again in three parts. Fix one part in Formin for morpho and freeze the other two parts for molecular analysis. The EVPS must be made of waterproof material that will not leach chemicals such as polymethyl acrylic, methyl polyoxyethylene, and steel.
The perfusion chamber must be at least 2.5 centimeters deep for minimal flexion and dilation of the vessel, and easy media changes. The vein support is a steel rod connected to L-shaped attachments from top to bottom. The connecting ducts on the pressure column allow pressure adjustments in and outflow to the vein and media change to determine the normal flow direction.
The two remaining segments for perfusion are gently injected with media. If the segment has valves, the flow will be slower. The vein must be reversed in the EVPS so that the valves don't hinder flow through.
Connect the vein to the two metallic cylinders. One end at a time. Secure the cylinders with Vicryl three oh around the indentations using a tight knot.
Then transfer the vein to the perfusion chamber loaded with media. For the third vein segment, attach one end of the vein to the cylinder as previously shown. Then gently slide the mesh on the vein.
Then secure the mesh and the vein on the cylinder with sutures. Finally, adjust the mesh length and secure the other end of the vein and the mesh to the cylinder. Move the assembly to the second perfusion chamber.
The EVPS is assembled in a closed loop with silicon tubing. Connect each outflow cylinder to a Y splitter using a segment of peroxide treated silicon tubing. Do the same for each inflow metal cylinder.
Next, connect the outflow splitter to a second Y splitter. From this Y splitter. Connect one outflow to the tube connected to the pressure transducer, and to connect the other outflow to the pressure column, use a one meter tube to connect the pressure column to the pump.
Complete the setup by connecting the pump to the inflow y splitter. With another meter of tubing, connect the remaining tubes to the pressure column. The lowest duct serves for media change.
After assembling the EVPS, fill the column with medium to just below the vein outflow duct. Now, move the system into the incubator, which is set to 37 degrees Celsius using carbon dioxide. Keep the pH at a constant 7.4 outside of the incubator.
Attach the pump gear heads to the gear drive to monitor the pressure. Connect the pressure output of the EVPS to the pressure transducer. Check that the tube is entirely filled and has no bubbles.
A pulses ital cardio signal at 60 pulses per minute with constant amplitude is set up via the computer software, which independently pilots the gearing pump. The resulting flow is about 160 milliliters per minute. The pressure is set up and regulated independently by a centrifugal pump in series with an adjustable pressure regulator controlled by the computer.
After three to seven days, finish the perfusion for each perfused segment. Remove five millimeters from each end to discard the ligated portion of the veins. Then cut a five millimeter ring for fixation from the central section of the vein.
Freeze the remaining fragments for molecular analysis. Embed the third segment in formin for histology and molecular analysis. After seven days of high pressure perfusion vein sections were prepared for histological analysis.
For all conditions, h and e staining revealed nuclei of endothelial cells lining the lumen and nuclei of SMCs in the media layer. When tissue was stained with V gel to view elastin perfused veins showed thickening of the intima pathological outward remodeling and media thinning was apparent. Masson's trichome staining shows that only one of three muscle layers persisted smooth muscle cells accumulated in the inner layer.
By contrast, the external reinforcement of the vein preserved SMC distribution and media structure This technique requires to work under rigorous sepsis. Once mastered, it can be performed in less than two hours using this system. Other conditions like turbulent flow can be tested in order to answer additional questions, such as the efficiency of the mesh reinforcement in non lanar flow.
