Inside-out and Standard Vein Grafts, with or without Muscle Filling, in Peripheral Nerve Repair: A Histomorphometric Study

Aim: The aim of this study was to compare two tubulization techniques, inside-out veins and standard veins, both filled with skeletal muscle or not, in sciatic nerve by morphological and histomorphometric study. Methodology: Seventy Wistar rats were divided in 4 experimental groups (IOVNF - inside-out vein with no filling; IOVSM - inside-out vein filled with skeletal muscle; SVNF - standard vein with no filling; and SVSM - standard vein filled with skeletal muscle) and a control group (Sham). The left external jugular vein was sectioned into about 14 mm segments to be used as autologous vein grafts. A 10 mm gap was then created in the sciatic nerve and the vein graft was inserted into the vein with or without filling of the right caudal tibial muscle. The animals were euthanized 12 weeks after surgery. Results: Myelinated and unmyelinated nerve fibers were observed in the histological analyses for all groups, as well as neoformation of the perineurium and intraneural organization of fascicles and blood vessels. In the morphometric analysis of the distal stump, regarding the myelin sheath area, all groups had a significant difference. The IOVNF group had the highest means for fiber, axon and myelin sheath areas. The SVSM group had the lowest means in all features measured, except for the axon area (4.95±1.72 graft; 3.71±0.90 distal stump). Conclusion: These results show that sciatic nerve repair with inside-out veins and no filling (IOVNF) had the best results, in the majority of measured variables, when compared to the other groups.


INTRODUCTION
Peripheral nerve lesions are frequently found in clinical practice and have great impact on morphofunctional alterations [1]. Despite continuous research and surgical innovation, peripheral nerve lesion repair with function recovery remains as a clinical challenge [2,3] that is influenced by several factors, such as: patient's age; and duration, degree and extent of the lesion [4].
Some studies have shown that tubulization techniques with standard and inside-out veins allow dissemination of neurotrophic factors, prevent the infiltration of fibrous tissue [15,16] and are composed of layers of laminin and collagen that are similar to the basal layers of nerve fibers, creating a microenvironment for axon growth [17]. However, other studies have shown that the tunica adventitia in inside-out vein grafts creates a more favorable environment for nerve repair [18][19][20][21].
The use of skeletal muscle as grafts in the tubulization technique started with the observation of the regular longitudinal alignment of muscle fibers [22,23], and the presence of extracellular matrix components that direct and increase the regeneration of nerve fibers [24][25][26]. Despite these advantages of using skeletal muscle as grafts for repairing nerve defects, few clinical studies have been reported to date [14]. Due to controversy in the literature about the use of standard or inside-out veins, the development of new research is necessary to contribute to this technique, which has great clinical applicability.
The importance of these methods of repairing peripheral nerve injury, and the small number of studies with morphometric analysis emphasizing their combination with muscle filling, led to the development of this study. The objective was to compare tubulization technique using inside-out veins (filled with skeletal muscle or not) with that using standard veins (filled with skeletal muscle or not) in the regeneration of the sciatic nerve, by means of morphological and histomorphometric analyses of graft sites and distal stumps. The animals were randomly divided into five groups: a Sham group (n=10) with no grafts, and four groups (n=15 each) submitted to a 10-mm gap in the right sciatic nerve and repaired with tubulization by means of a graft taken from the left external jugular vein filled with skeletal muscle or not.

Animals and Surgical Procedures
Animals were first weighed and then anesthetized by intramuscular injection of 50% tiletamine and 50% zolazepam at 10 mg/kg (Telazol ® ; Fort Dodge Laboratories, USA). Aseptic techniques were adopted in all surgical procedures involving the animals.
All surgical procedures were performed with the help of a stereo microscope (MC/M9 -DF Vasconcellos S/A, Sâo Paulo, Brazil). After anesthesia and trichotomy on the left side of the neck region, a 20-mm longitudinal incision was made in the skin. The jugular vein was isolated and ligature of its confluents was done; then a periodontal probe was introduced and both ends were sectioned (Fig. 1a). In Groups IOVNF and IOVSM, vein was reversed with the aid of a periodontal probe. The option to use the external jugular vein was due to its larger diameter and the absence of valves, which presented fewer obstacles to axonal regeneration [18].
This segment of the vein had an average diameter of 1.6 mm to 2.1 mm and was 14 mm in length [27], and was kept in physiological serum until its placement, as a graft, between the injured nerve stumps. The skin of the donating site was sutured with 4-0 monofilament nylon thread (Ethicon Inc., Somerville, NJ, USA). In the second step, the sciatic nerve (approximately 1.3 mm in diameter) was exposed (Fig. 1b) and sectioned at the right side, and a 10-mm segment was removed. The Sham group got only an incision for exposure of the right sciatic nerve, without injury; the IOVNF group (inside-out vein without filling) got a graft from the left external jugular vein inside-out; the IOVSM group (insideout vein filled with skeletal muscle) got a graft from the left external jugular vein with a sample of right caudal tibial muscle (Figs. 1c and 1d); the SVNF group (standard vein without filling) got a graft from the jugular vein in standard position ( Fig. 1e); the SVSM group (standard vein with skeletal muscle) got a graft from the jugular vein in standard position with right caudal tibial muscle.
A segment of right caudal tibial muscle approximately 10 mm in length, with a diameter similar to the external jugular vein, was introduced into the venous lumen in groups IOVSM and SVSM [28].
The epineurium was sutured with 10-0 monofilament nylon suture (Ethicon Inc., Somerville, NJ, USA) ensuring that both the proximal and distal stumps went inside the inside-out and standard vein grafts, about 2 mm at each end. Subsequently, the skin was sutured with 4-0 monofilament nylon suture (Ethicon Inc., Somerville, NJ, USA).

Euthanasia and Collection of Nerve Segments
Euthanasia of all animals was done after 12 weeks by means of an intraperitoneal injection of a high dose of pentobarbital (Nembutal ® , 30-50 mg/kg, Abbott Laboratory, Quebec, Canada). Afterwards, two fragments were collected, one from the middle third of the vein graft, and the other from the middle third of the distal stump of the right sciatic nerve (Fig. 1f). After this period of fixation, the sciatic nerves were sectioned crosswise into two segments and left in the same fixative solution that was previously used for a period of one to two hours, and they were post-fixed for two hours in osmium tetroxide at 1% [29] in sodium cacodylate buffer 0.1 M pH 7.3. Subsequently, they were dehydrated in ethanol and propylene oxide and embedded in resin for histological examination. Cross sections of 0.5 µm were obtained with a microtome (RM2265, Leica Biosystem ® , Germany), colored with toluidine blue at 1% in borax aqueous solution at 1% and osmium tetroxide for morphological and histomorphometric analysis.

Histological Processing of Sciatic Nerve Samples
The images of histological sections of grafts and distal stumps were captured with 400 times magnification (40x lens), by means of a video camera coupled to an optical microscope (Axiophot 2 Zeiss KS -300, Germany) and a computer.

Morphological and Histomorphometric Analyses
In the analysis of the sciatic nerves in the experimental groups, the neuronal morphology and the entire set of structures were observed, namely, the epineurium, perineurium, adipose tissue, intraneural and extraneural blood vessels and myelin sheath.
For the Sham group, assessment of the area and minimum diameter of nerve fibers was performed, as well as the area and minimum diameter of axons, the area and thickness of the myelin sheath; for the other four groups (IOVNF, IOVSM, SVNF and SVSM), the assessment was made on the graft and the distal stump. These measures were obtained in four randomly selected fields by using the Image-Pro Plus™ program, version 6.0 (Media Cybernetics, Rockville, MD, USA). For the histological slides, the assessment was made with SigmaScan Pro version 5.0 (San Jose, CA, USA).

Statistical Analysis
Data obtained were submitted to ANOVA, followed by Scheffé's test. For all analyses, p<0.05 values were considered as statistically significant.

RESULTS
Myelinated and unmyelinated nerve fibers were observed in the histological analyses of the middle third of the grafts (Fig. 2) and the middle third of the distal stumps (Fig. 3), for all groups, as well as neoformation of the perineurium and intraneural organization of fascicles and blood vessels. The pattern of constitution of nerve fibers in most grafts of the experimental groups was similar to that of the sciatic nerve of the Sham group.
It was possible to observe the axons being regenerated and placed outside the epineurium on the site of the grafts (Fig. 2) and the distal stump (Fig. 3) in all experimental groups. It was also possible to see the thickening of the perineurium within the experimental groups when compared to the Sham group.
In the IOVSM and SVSM groups, on the graft site (Fig. 2), the nerve fibers appeared in wellorganized microfascicles and the presence of remains of muscle fibers.

DISCUSSION
The objective of this study was to compare the tubulization technique using inside-out veins (filled with skeletal muscle or not) to that using standard veins (filled with skeletal muscle or not) in the regeneration of the sciatic nerve. Our results indicate that sciatic nerve repair with inside-out vein grafts and no filling (IOVNF) has the best results, in the majority of measured variables, when compared to the other experimental groups.
In recent decades, much progress and innovation have taken place in techniques of surgical repair of injured peripheral nerves aiming to reestablish their function [2,3,6,30]. In this study, it was possible to observe the axons being regenerated and placed outside the epineurium in all experimental groups. Vein grafts form structures that favor the migration of axons to distal stumps and provide a favorable microenvironment of extracellular matrix and growth factors for the maturation of cells [31][32][33]. This phenomenon occurs because vein grafts are rich in collagen and laminin to better guide axonal growth to the distal end of the injured nerve and have lower inflammatory response [34][35][36].
The absence of muscle filling in the vein graft can make the vessel collapse, due to the action of adjacent tissues [37][38][39]. This occurs in gaps that are wider than 5 cm [40,41] In contrast, the IOVSM and SVSM groups had muscle fragments at the graft site and the lowest means for the analyzed variables. We tested the hypothesis that the use of muscle fibers could increase axonal sprouting, due to availability of a longitudinally oriented basal lamina and extracellular matrix components [6,22], however the presence of muscle resulted in disorganized growth, and could even make it difficult for axons to pass through the graft.
Of all the variables analyzed, on the graft site, there was a significant difference between the IOVNF group and the other groups in the variables: area and fiber diameter, axon diameter and thickness of the myelin sheath, confirming that the inversion of the vein in the tubulization technique contributed to nerve regeneration through the microenvironment, which was high in collagen, lamini, and the presence of Schwann cells in the tunica adventitia [20,44].
The SVSM group had the lowest means, due to the probable resistance of the muscle to the growth of axons and the absence of the tunica adventitia [6]. The application of this knowledge for routine clinical practice are based on studies showing that the venous grafts, combined or not with muscle tissue in clinical cases, usually have good results, improvement of up to 85% of our cases with a minimum follow -up of 14 months [45].
There were some limitations on this study. We could have performed the functional tests and use of post-surgery techniques for evaluating possible functional improvement, such as laser therapy [46], electrotherapy [47], or physical exercise [48]. However, this study was focused on better understanding of the recovery of the peripheral nerve using two grafting techniques with veins filled or not with muscle, focusing on the morphometry of the nerve fibers.

CONCLUSION
Based on the results of the present study, we concluded that sciatic nerve repair performed by the technique of inside-out veins and no filling had the best results (IOVNF group), in the majority of measured variables, when compared to the other experimental groups.

CONSENT
It is not applicable.

ETHICAL APPROVAL
The study was approved by the ethics committee of the University of Marília (Marília, São Paulo, Brazil).