An Analysis of Repair by using a nNerve cConnector as an aAlternative for dDirect rRepair in pPeripheral nNerve iInjury

Introduction

Nerve injuries can impose a heavy burden on patients' quality of life. AltThough direct epineurialum and perineurialum sutures are the classic techniques most commonly used for peripheral nerve injury, they are often accompanied with by undesirable tension and their surgical outcomes are can be less than satisfactory.^{(1-)(2)(3)(4)} Such could be due toThese sutures can cause axon misalignments (such as miss direction, overlapping, and buckling) of axons , ingrowth of scar tissue ingrowth into the repair site, and or tension-induced ischemia around the stumps, all of which can leading to poor fascicular coaptation.⁽⁵⁾ To overcome these problems, several newer technologies methods, such as connector repair, have been considered.⁽⁶⁾

Previous studies have suggested that utilizing using a nerve conduit as a connector (covering the repair site and leaving a very short gap between the stumps) for peripheral nerve repair, covering the repair site and leaving a very short gap between the stumps, provides outcomes that are equivalent to, or better than, those of direct suture repair.^(7-)(8)(9) It isNerves generally considered that nerves regenerate along the length of the nerve conduit in a selective manner, such that thewith proximal motor nerves migratinge to join the distal motor nerves.^(10,)(11) The intentional gap in the connector is known to provides an area for this selective reinnervation, thus encouraging natural re-growth while preventing the aforementioned misalignments.

The cConnector also provides a physical barrier to preventing the infiltration of scar tissue, in effect allowing the diffusion of nutrients to the healing repair site.^(12,)(13) Thise barrier prevents the any potential uncontrolled sprouting to the outside of the coaptation site,⁽¹²⁾ while and itsthe additional length relocates the sutures away from the coaptation site, thus reducing the risk of tension and tension-induced ischemia around the stumps. The use of a connectors also reduces decreases the number of sutures required for the nerve coaptation, which potentially may reduces surgical time.^(6,)(12)

While the employmentThe use of a nerve conduit as a nerve connector has been known to reportedly achieved outcomes equivalent to, or better than, those produced by direct suture repair; ,however, its the underlying mechanisms of the conduit and how the response of thethe axons behave in the conduitit still remain unclear. In tThe present study, we investigates the peripheral nerve repair mechanisms inside of an artificial nerve conduit through across a short gap. To do this, we performed Sserial in vivo imaging using of transgenic mice which that constitutively express yellow fluorescent protein (YFP), fluorescence and analyzedsis of gene expressions at the repair, were conducted while and recorded various histomorphometric were taken. The results wereWe compared all conduit repair findings against their direct suture repair counter-parts and against the control. FThe final recovery was quantified through functional assessments.

Materials and Methods

Preparation of nerve conduit

For the present study, we used a bBioabsorbable polyglycolic acid (PGA) conduit filled with collagen in a honeycomb-like structure (Nerbridge, Toyobo Co. Ltd.; Fig. 1A); used in this study are this product is commercially available and hasve been approved by the Japanese government as a medical device for clinical practice by the Japanese Government since 2013. We selected Tthis artificial nerve was selected for this study for its robustness. The tube was made of PGA fiber mesh, that was permeable for to particles smaller than 600 KD., with theIt had an inner diameter of 1 mm and the a length of 55 ± 5 mm, and was designed to biodegrade 3 months after transplantation in vivo.⁽¹⁴⁾ For the presentthis study, only the outer cylinder without the inner honeycomb--shaped collagen was used,⁽¹⁴⁾ after being soakeding in saline for 5 minutes.⁽¹⁵⁾

Experimental Aanimals

In this study, We purchased 64 transgenic mice (Thy1 -yellow fluorescent protein ( YFP) 16), available from tThe Jackson Laboratory, ( strain : B6.Cg-Tg[Thy1-YFP]16Jrs/J; 8–12 weeks old, weighing 23–28 g, 32 males and 32 females; Fig. 1B–DB6. Cg-Tg (Thy1-YFP) 16 Jrs/J) . In these mice, with all axon fibers of the motor and sensory nerves constitutively express ing YFP fluorescence. were employed (8-12 weeks old, weighing 23-28 g, 32 males and 32 females, Fig. 1B, C and D). The aAnimal husbandry was conducted in accordance to with the standards and regulations provided by the National Institutes of Health Guide for Care and Use of Laboratory Animals. The study was approved by the Animal Care Committee of Juntendo University. The mMice were kept in groups of four in a sterile cages that were maintained at 22℃ ± 2℃ and 40-60% humidity under a 12-hour light/dark cycle. The mice, and they had free access to water and 15- kGy gamma-irradiated feed (CRF-1, Oriental Yeast Co.). Animals They were sacrificed by cervical dislocation under deep anesthesia after the all grafts were had been harvested.

Experimental model

The experimental group (n = 48) underwent a nerve transection procedure to create an injury toon both the sciatic nerve of each hind limbs. which were laterTwo weeks later, this was followed by two types of repair operations:, direct epineurialum suture (right hind limb) and artificial nerve connector (left hind -limb). The control group (n = 16) underwent the same procedures but without the nerve transection or the repair operation. Equal numbers of male and female and male mice were assigned to each group.

-Creation of Ssciatic nerve injury model creation

The mice in the Eexperimental group were anesthetized with inhalation anesthesia ofvia inhaled sevoflurane (5% sevoflurane for induction, 2% for maintenance). On each hind limb, Aa 15 mm skin incision was made parallel to the femur on the both hind limbs to expose the sciatic nerves. For To manipulateion of the sciatic nerve, a microscope was used (MZ16FA, Leica Microsystems) was used. Once the nerve was detached from the surrounding tissues, it was transected at the halfway between the hip and knee joint (Fig. 1E–, F and G). The Ffemoral muscle was partially peeled and placed between the nerve stumps to avoid natural reconnection, and the wound was closed using 5-0 nylon. The animals were then left for 2 weeks for (to allow the formation of Wallerian degeneration in the distal stump) before the repair operations were conducted performed.

-Repair operations

The experimental group underwent the repair operations two 2 weeks after the initial lesion-generating operation. Each mouse was placed Uunder anesthesia, the then its wounds wereas reopened, and the sciatic nerve stumps were exposed under the a microscope (Fig. 1H and I). The formation of Wallerian degeneration was confirmed through by the disappearance of YFP fluorescence expression in the distal stump (Fig. 1J and K). The ends of both stumps were debrided to the minimum extent in order to remove the neuromas, and the two types of nerve repair (one to each hind limb) were performed to each hind limbas follows.

Epineurialum suture –- Dddirect repair (DR): 

The right hind limb of each each experimental mouseTo every right hindlimb was repaired using, a direct epineurialum suture, which was conducted with two stitches of 9-0 nylon stitches (Fig. 2A –, B and C).

Nerve repair using nerve connector –- Cconnector repair (CR): 

Every left hindlimbThe left hind limb of each experimental mouse was repaired using a 3 mm artificial nerve. The artificial nerve was interposed with a stitch of 9-0 nylon on either each end. Approximately 1 mm of the each nerve -ends were was inserted into the conduit, leaving a distance of 1 mm between the stumps inside the conduit (Fig. 2A, D, and E).

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

To analyze the gene expressions at the repair site, in the case of DR, tissues between 1 mm proximal and 1 mm distal from the suture (in the case of DR), and in the case of CR, or all tissues inside the conduit (in the case of CR), wasere harvested and examined by subjected to qRT-PCR at 3 days, 1 week, and 2 weeks after the repair operation (n = 24). The eExpressions levels of a macrophage migration marker (CD68), an angiogenesis marker (vascular endothelial growth factor ([VEGF)]), an endothelial marker (platelet endothelial cell adhesion molecule CD31[(PECAM-1)], also known as CD31), Schwan cell migration markers (SOX10, and S100), and neurotrophins (neurotrophin-3 [(NTF-3)] , brain-derived neurotrophic factor [(BDNF)], and nerve growth factor [(NGF)]) were analyzed. The tissues were was extracted and stored in RNAlater solution at −-4°C overnight, and then stored at −-80°C until RNA extraction. Total RNA was extracted with using an RNeasy Plus Universal Mini Kkit (QIAGEN), and RNA purity and concentration were assessed using a NanoPhotometer (IMPLEN). RNA samples with a 260 nm/280 nm ratio between 1.8 and 2.0 were used for qRT-PCR. We used a high-capacity RNA-to-cDNA kit (Applied Biosystems) for reverse transcription into cDNA and TaqMan Fast Advanced Master Mix (Applied Biosystems) for qRT-PCR. All primers were purchased from Applied Biosystems, and the measured values were compared with using GAPDH as a reference gene. An ABI 7500 FAST PCR system (Applied Biosystems) was used for The thermocycling, and the conditions were 95°C for 20 seconds , followed by 45 cycles of 95°C for 3 seconds and 62°C for 30 seconds, on an ABI 7500 FAST PCR system (Applied Biosystems). The amount of each target gene relative to the GAPDH reference gene was determined using the comparative threshold cycle method, and the mean values were calculated.