The embracing end-to-side neurorrhaphy in rats 1 A neurorrafia término-lateral abraçante em ratos

PURPOSE: Compare two new methods with the traditional end-to-side neurorrhaphy. METHODS: Rats were divided into four groups. In A-L group the peroneal nerve was sectioned and the distal stump was connected to the lateral of the tibial nerve (donor) with two 10-0 nylon points. In A-R group two perineurium flaps embraced the donor nerve. In the B-R group a suture embraced the donor nerve. Group B-L was the control. After six months tibial cranial muscle mass and morphometry of the distal stump of the peroneal nerve were evaluated. RESULTS: Muscle mass in groups A-R, A-L and B-R were lower than B-L group (p<0.0001) an equal between themselves (p>0.05). Groups A-R, B-R and A-L had a lower number of nerve fibers when compared with B-L (p=0.0155, p=0.016, p=0.0021). CONCLUSION: The three types of neurorrhaphy showed no differences related to muscle mass and number of nerve fibers suggesting that the embracing with a single suture has great potential due its simplicity and usefulness in deep areas.

When there is the proximal and distal stump after a nerve section it is possible to perform the surgical repair with the Endto-End Neurorraphy (ETE).In this process there is the union of the endoneurium conduits, making the nerve fibers regeneration easier 6.In many clinical situations the proximal stump is not available, making the end-to-end neurorraphy impossible.In these cases, an option is the end-to-side neurorraphy, introduced by Viterbo et al. 1 , without lesion of the donor nerve.In experimental studies in rats, the peroneal nerve was sectioned and its distal stump was sutured to the lateral face of the intact tibial nerve, with and without the epi-perineurium removal.The proximal stump was put away and buried in the subjacent muscle.It was noticed growth of the axons from an intact nerve into the distal stump of a receptor nerve.For the first time they obtained muscle reinnervation without lesion of the donor nerve.The importance of this study was that any nerve can be a potential axonal donor.Later studies proved these initial findings 7,8 .
Today we know that after some months of ETS, there is the absorption of the conjunctiva layers and lateral sprouting of axons coming from the intact nerve to inside of the receptor nerve, maybe due to the liberation of enzymes or nerve growth factors, still unknown.The direct contact between the donor and receptor nerve seems to be a prerequisite to the reinnervation, because it allows the interaction of the Schwann cells between the two nerves, maybe increasing the production of growth factors, inducing and leading to the axonial sprouting inside the sectioned nerve 9 .
Currently the ETS is performed with two to four suture stitches in the lateral surface of the donor nerve.This is time consuming and a more delicate procedure.In an attempt of simplify this technique, two simple methods were tested, the "embracing" with epineurium flaps or just one "embracing" stitche.
The aim of this study was to compare two new methods to perform the ETS with the traditional one.

Methods
Twenty Wistar rats were divided between two groups, A and B. Each side was considered a sub-group, right (R) and left (L), constituting the groups A-R, A-L, B-R and B-L.
The rats had their mass measured and anesthetized with ketamine and xylazine at a dose of 70 mg/kg and 30 mg/ kg respectively, intramuscularly.The posterior members had a 2 to 3 centimeters longitudinal incision, compromising skin and subcutaneous with posterior section of the biceps femoris muscle.After that, the procedure was performed, according to the experimental group.
In the group A-L a traditional end-to-side neurorrhaphy (TETS) was performed.The peroneal nerve was sectioned and the distal stump was sutured to the lateral surface of the tibial nerve with two 10-0 nylon stitches (Figure 1).In the group A-R an embracing end-to-side neurorrhaphy with epi-perimeurium flaps (ETSF) was done.The extremity of the distal stump of the peroneal nerve was divided longitudinally in two parts and the axons were removed, resulting in two epiperineural flaps.These flaps surrounded the tibial nerve and were fixed with one 10-0 nylon stitche (Figure 2).In the group B-R an embracing end-to-side neurorrhaphy was performed with one embracing 10-0 nylon stitche (ETSS).The procedure was almost the same than in the group A-R, although without the epineurum flaps.A single nylon 10-0 point went through the epineurum of the peroneal nerve, passed behind the tibial nerve, came back and passed in the front of this nerve, and then went through the epineurum of the peroneal nerve again like a loop, in a way that when fixed, this point approximated the extremity of the peroneal nerve distal stump to the tibial nerve (Figure 3).The group B-L (Control) was the normal control and did not received any procedure (Figure 4).The epineurum of the tibial nerve were intact in all groups.The proximal stump of the peroneal nerve was inverted, buried in the subjacent muscle and fixed with one 5-0 nylon sticthe.
The incision was sutured by plans with nylon 5-0.
The animals were housed in appropriate cages of the Animal Laboratory of Surgical Technique and Experimental Surgery, Department of Surgery and Orthopedics, with temperature of 24 degrees (+/-0.5),with water and ration ad libitum, respecting the 12 hours dark/clear cycle.
After six months the rats were sacrificed and the cranial tibial muscle and the distal stump of the peroneal nerve were harvested.The muscles had their mass measured and the nerves were submitted to fibers counting.
For statistic analysis, since the variable answers did not show adherence to the normal distribution of probabilities, the Mann-Whitney non-parametric technique was used.All the comparisons were performed with the significance level of 5%.

Results
Table 1 and Figure 5 refers to the average of the cranial tibial muscle mass.All groups were different from the control but not between themselves.

Discussion
In 1895 Ballance (apud Ballance et al. 10 ) performed for the first time the suture of sectioned nerve to the lateral of a donor nerve, after cutting partially the donor.These lesions, determined by incision 10 or by scarification 11 in the lateral aspect of the donor nerve, allow the sectioned axons to grow inside the receptor nerves, providing, in fact, an end-to-end union between the endoneural tubes from both nerves.
These methods invariably showed functional disadvantage to the muscles innervated by the donor nerves.This observation leads Babcock 12 to suggest the definitive abandon of the neurorhaphy.After that, except for the Gatta 13 and Pozzan 14 publications, there was a gap in the literature, without any report about this subject.For these reasons plus the operative microscope and microsurgical techniques advent, the end-to-end neurorraphy (ETE) became the standard treatment for peripheral nerve lesion, and the end-to-side neurorraphy (ETS) was forgotten 6 .
In 1992 [1][2][3] , with the introduction of the new concept of the end-to-side neurorraphy (ETS) without lesion on the donor nerve, new surgical possibilities appeared.Since the donor nerve doesn't suffer any lesion, any nerve can be used as donor nerve.
The fibrin glue, already used in the ETE [19][20][21][22][23] , can also be used in the ETS 4 .An important disadvantage of this method is it high cost 24 .However it's use became very interesting, especially in some situations where the nerve to be repaired are deep, making it more difficult to do the sutures.
In this study it was tried a most simple alternative, that could also be applied in the depth.The result of this study showed that both proposed new techniques, epineurium flap and embracing suture were similar to the traditional ETS.
In the embracing suture it is obvious that the suture can't be neither very tight, because it would cause stenosis in the donor nerve, nor very floppy, because it wouldn't provide appropriate contact between both nerves.However the ideal tension still was not determined and it's a subjective factor.
An interesting and contradictory aspect is the opinion of some authors who advocates lesion on the donor nerve, even minimum, like the ones caused by the needle during the suture in the end-to-side neurorraphy 5,[25][26][27] .These small lesions would be important to provide bigger contact between the nerves and also for the liberation of growth and neurotrophic factors, related to the development, maintenance and regeneration of the axonal sprouts 28 .For this reason and also to reduce the dehiscence, some authors advocate the association of some anchorage microsurgical points in the ETS with fibrine glue 29 .
In the present study no donor nerve lesion was done in both embracing ETS groups, and the axonal sprouting occurred similarly to the traditional ETS, where needle was used.Other authors, like Akeda et al. 30 an Matsumoto et al. 31 also noticed axonal regeneration with no donor nerve lesion, using a silicone chamber in "T" and without suture.These authors understand that the minimum lesion caused in the donor nerve can harm it, so it must be avoided 5,31 .
The results obtained in the ETS with epi-perineural embracing flap and ETS with embracing suture support the theory that the two proposed techniques can be used in the ETS, even without harming the donor nerve.
The axonal sprouting exact mechanism through ETS still was not established 5,30,32 .The macrophages, related to the Walerian regeneration, seem to be the responsible for the perineurum absorption, allowing the neurotrophins from the receptor nerve to initiate the axonal sprouting 5,33 .This would explain the axonal sprouting after the embracing ETS, even without harming the donor nerve.
Liu et al. 5 questioned the reproducibility of experimental studies in human begins.The authors affirm that the bigger perineurium, epineurium e endoneurium thickness from the human begins could prevent the axonal sprouting with techniques that doesn't cut the donor nerve.However, Samii et al. 32 reported success in a clinical case using ETS with fibrin glue, that, in the same way than the embracing from this study, didn't have any Many experimental and clinical studies met good results with the ETS 9,10,[34][35][36][37] .Some, however, were not success or the results were limited 4,[38][39][40] .Several technical aspects in the ETS performance are being studied and maybe can explain the bad results found by some authors.
In the present study was proved that the similar results between the three types of ETS, traditional, embracing with a single suture or two epineurum flaps.This leads us to believe in the big potential of the embracing ETS with a single suture because it's simpler to be done, especially in deeper places.

Conclusion
The three types of neurorrhaphy showed no differences related to muscle mass and number of nerve fibers suggesting that the embracing with a single suture has great potential due its simplicity and usefulness in deep areas.

FIGURE 1 -
FIGURE 1 -Traditional end to side neurorrhaphy as described by Viterbo et al. 1 .

FIGURE 4 -
FIGURE 4 -Normal control group did not received any procedure.Sciatic nerve and its three branches: peroneal, tibial and sural nerve.

FIGURE 5 -
FIGURE 5 -Mean and standard deviation of the cranial tibial muscle mass.The groups ETSF, TETS and ETSS exhibited fewer nerve fibers when compared to the Control Group (p=0.016).

TABLE 1 -
Mean and standard deviation of the cranial tibial muscle mass.

Table 2 and
Figure 6 refers to the average number of nerve fibers per microscopic field of 0.175 mm 2 .The experimental groups also showed lower results from the control group, but not between themselves.

TABLE 2 -
Mean and standard deviation of fibers number per microscopic field.