Tips and Tricks in Experimental Nerve Defect Surgery

Microsurgery in itself might be considered a challenge for some plastic surgeons, as it requires patience and perseverance. Furthermore, when it comes to nerve surgery, the challenge is even greater as it implies a longer time necessary to observe results. In clinical practice, a good technique is associated with a good outcome. However, the problem of the recovery time remains an issue to be addressed by future generations. Nerve defects represent an even greater challenge in traumatology. Crush injuries, lacerations and avulsions may all lead to irreversible damage of the nerves. In such cases, special surgery techniques are required to restore function1-4. In order to prepare for these special techniques, a surgeon must begin with experimental in vivo nerve surgery and one that has the highest similarity with humans is the sciatic nerve of the Wistar rat5.


INTRODUCTION
Although quite resistant to shear stress, nerves can be injured in diff erent ways at diff erent sites 6,7 . Nerve defects up to 0,5cm can be sutured sometimes primarily, with proximal and distal dissection. However this results in higher tension in the suture, increasing the risk of nerve rupture and infl uencing the regeneration process. For this situation, coaptation of the nerve

MATERIAL AND METHOD
Wistar rats in diff erent microsurgery training laboratories were used. Th e weight of the rats varied between 250-350g being of diff erent ages (7 to 9 weeks). Th e anesthesia protocol consisted of a mixture of ketamine (75mg/kg) and xylazine (10mg/kg) administered intraperitoneally. For a correct dose administration, a scale was used to weigh every animal. In all laboratories, diff erent microscopes with magnifi cation varying between 3x-20x were used, as well as suitable microsurgical instruments appropriate sutures (8-0.9-0.10-0).
After administrating the anesthesia, the rats were shaved in the gluteal area and the thigh region and pla-One of the most suitable laboratory animals for in vivo experimentation is the Wistar rat, due to its high similitude in nerve regeneration; furthermore its availability ease for manipulation make the rat the ideal candidate for experimental microsurgery. What is more, the sciatic nerve in the case of the Wistar rat is easily accessible and has suitable size and length for diff erent microsurgical procedures 14 . Th e sciatic nerve runs along the inferior border of the femur for 3cm before dividing into 3 terminal branches -common peroneal, tibial and sural nerve 15 .
Apart from the surgical technique, good outcomes in the in vivo experiments depend on proper anesthesia 16 .

Wistar rats
Microsurgical instruments ced in prone position, with the limb to be operated on in extension. A syringe was placed under the hip joint in order to achieve maximal limb extension and to push the sciatic nerve more superfi cial. An oblique-transverse skin incision was made 1cm inferior to the border of the femur and dissection through the biceps muscle fi bers was performed in order to expose the underlying sciatic nerve.
After creating a 0.5cm nerve defect, a surgical repair using diff erent procedures was performed. Some involved using a silicone tube, others a biologic vessel harvested from a rat. Both procedures require placing the nerve endings inside the conduit for 0.3cm and 2-3 sutures at both ends.

RESULTS
In all cases when a defect is involved, the nerve must grow through 2 diff erent sites -proximal and distal. Th e outcome after nerve repair depends on the technique used, on the type of nerve conduit as well as on the type of the initial nerve injury. Th e regeneration speed of the sciatic nerve in rats is estimated at 1,5mm/day and fi nal results may be observed several weeks after the intervention. Th is growth rate may be increased by using diff erent growth factors inside the nerve conduits 17 .

DISCUSSIONS
Choosing adequate size rats is important in order to have large enough nerves to practice the microsurgical technique. Th erefore, it is recommended not to operate rats which are lighter than 250g or younger than 7 weeks of age.
Th e exact dosage of anesthesia is vital to be administered in order for the animals to survive and to have a fast recovery (if a longer study is in question). When the correct dosage doesn't have the desired eff ect, it is better to postpone the operation and choose another rat if possible (multiple doses may cause respiratory distress which can fi nally kill the lab animal).
Synthetic nerve tubes represent a good therapeutic option for nerve defects. Th ey do not require a prior intervention for harvesting; their lumen is generally open (no external growth resistance) and can be fi lled with diff erent growth factors. Th ey can be manufactured to Sciatic nerve exposure Sciatic nerve repair using a silicone tube Sciatic nerve repair using aorta as nerve conduit (arrow -aorta; * -resected nerve to create the defect) sizes. Th e major problem with veins is that they tend to collapse, making it diffi cult for the nerve to grow to reach the distal ending. A solution for this issue is to fi ll their lumen with either muscle fi bers or diff erent growth factors, in order to keep the lumen open 18 . Manoli et al showed no diff erence between nerve autografting and muscle in vein conduits for defects of the digital nerves 19 .
Arteries can also be used as nerve conduits. Th ey are scarcer to fi nd and when harvested may result in some degree of functio laesa but they have the advantage of a relative rigid wall, keeping the lumen open. Th erefore they can be more easily fi lled with semiliquid substances (nerve factors) that remain inside. Th e diff erence in caliber in comparison to the nerve may determine notching of the vessel. To prevent this, equally distanced sutures on the circumference of the vessel should be performed.
In case of a small caliber artery to be used as a nerve conduit, this can be adjusted by making fi shmouth incisions in order to fi t the nerve endings inside the lumen.
Although nerve conduits may be used to bridge a nerve gap and facilitate grow-through process, autografts/allografts have similar result and are frequently used in nerve defects; there are however pitfalls in using either methods, which need to be kept in mind when dealing with nerve defects 20,21 .

CONCLUSIONS
Nerve surgery requires special microsurgical training. Experimental microsurgery performed on living rats off er a real experience when it comes to handling life tissue. Nerve defects can be solve only by using diff erent types of nerve conduits, each having advantages and disadvantages. be suitable for every nerve size. Th e disadvantages of synthetic tubes are that they are expensive and they might generate a foreign body reaction. Furthermore, their structure can be rigid and when they don't have the right size, they may not be adjusted to nerve diameter.
In comparison to synthetic tubes, vessel conduits are easily accessible and abundant. When harvested from the same rat, these generate little infl ammation, but no foreign body reaction. Due to the elasticity of their walls, veins can be adjusted to fi t diff erent nerve Notching and twisting of the aortic conduit due to improper placement of the sutures Small caliber artery adjusted by a fi shmouth incision (arrow) to fi t the nerve inside the lumen Aortic conduit harvested from another rat (notice the larger caliber of the conduit in comparison to the nerve). Suture at regular intervals prevent the notching Knowing the shortcomings of each conduit (biological or synthetic) and what can be done in each situation is the prerogative to performing a nerve repair with good expected outcomes.
Compliance with ethics requirements: Th e authors declare no confl ict of interest regarding this article. Th e authors declare that all the procedures and experiments of this study respect the ethical standards in the Helsinki Declaration of 1975, as revised in 2008(5), as well as the national law. Informed consent was obtained from all the patients included in the study.