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The purpose of this study was to clarify the effectiveness of hyaluronic acid (HA) in the prevention of scar formation after neurolysis using a rabbit model. In the first stage, the left sciatic nerve was exposed and elevated along a 3 cm section. Then, the surface of the neural bed was coagulated using a bipolar coagulator. Finally, the sciatic nerve was replaced and fixed to the neural bed with 8/0 nylon sutures, and the wound was closed. In the second stage, the adherent sciatic nerve was re-exposed after 6 weeks. In the neurolysis group, a simple neurolysis was performed. In the HA group, the neurolysis was performed in a surgical field coated with HA from the beginning to the end of the operation. In the steroid group, methyl prednisolone acetate was infiltrated at the end of the neurolysis. In the third stage, electrophysiological, histological and biomechanical measurements were taken 6 weeks after the second stage. While there was no significant difference between the HA and the steroid groups, the electrophysiological functions of the HA and steroid groups were significantly better than that of the neurolysis group. Histology showed that the formation of intraneural and extraneural scar tissue was lowest in the HA group, followed by the steroid and neurolysis groups. The tensile strength required to strip the sciatic nerve from the neural bed of the HA group was significantly less than that of the neurolysis group. However, there was no significant difference between the steroid and neurolysis groups. In conclusion, HA effectively reduced scar formation after neurolysis.
One of the major problems after peripheral nerve surgery is scar formation between the nerve and the neural bed. Extraneural scarring causes nerve compression, tethering and vascular compromise and, over time, can also lead to intraneural scarring.
Since postoperative adhesion and extraneural or intraneural scarring create persistent or new peripheral nerve symptoms, adhesion is receiving attention as an important complication of surgery. While many strategies have been used in an attempt to prevent adhesion in experimental studies,
results have been poor. Hyaluronic acid (HA) is easy to apply in the surgical field because it is a commercially available sterilised solution. Since HA may prevent intraperitoneal adhesion,
using HA during neurolysis may reduce extraneural and intraneural scarring. The purpose of this study was to clarify the effectiveness of HA in preventing adhesion after neurolysis.
1. Materials and methods
1.1 Experiment 1
Japanese white rabbits weighing 2.5–2.7 kg were anaesthetised by intravenous injection of sodium pentobarbital (30–50 mg/kg). The surgery was conducted in three stages. Stage 1 produced adhesion, stage II attempted neurolysis with or without drugs and stage III estimated the grade of adhesion. The control group consisted of the nonoperated intact right sciatic nerves.
In stage 1, the left sciatic nerve was exposed at the mid-thigh level and elevated over a length of 3 cm using a sterile technique. Adhesion was produced according to Abe et al's technique.
The entire surface of the neural bed, which mainly consisted of semi-membranous and adductor muscles, was coagulated using a bipolar coagulator (Fig. 1(A)) . Then, two epineural sutures were used to fix the sciatic nerve to the coagulated neural bed with two 8/0 nylon threads. It was necessary to fix the nerve to the neural bed because it was difficult to establish the adhesion without fixation.
After this, the wound was closed, and scar tissue formed around the sciatic nerve over time.
Fig. 1Macroscopic findings in the experimental model. (A) In the first stage, the rabbit's sciatic nerve is elevated and the neural bed is coagulated. (B) In the second stage, marked adhesion between the nerve and the neural bed is observed (adhesion group).
In the second stage, 6 weeks after the first stage, the sciatic nerve was re-exposed (Fig. 1(B)). In the adhesion group, adhesion was completed. In the neurolysis group, neurolysis and cutting of the two-epineural sutures were performed without using any drugs. In the HA group, the sciatic nerve was coated in 3 ml of HA during the entire neurolysis procedure. HA coating of the surrounding tissues was performed throughout the surgical period to ensure that the wounds were in contact with HA continuously. HA was already sterilised and packed in a syringe, marketed for treatment of osteoarthritis of the knee joint (Arzt®, Seikagaku Corporation, Japan). The molecular weight of the HA was 84×104.
The neurolysis took approximately 20 min, and the nerve was coated with HA during this period. In the steroid group, the steroid solution (5 mg of methyl prednisolone acetate (Depomedrol)) was infiltrated at the end of the neurolysis. This is a common procedure in clinical practice.
In the third stage, 6 weeks after the second stage, the sciatic nerve was re-exposed, and electrophysiological, histological and biomechanical studies were performed.
1.1.1 Electrophysiological evaluation
The compound muscle action potential (CMAP) of the tibialis anterior muscle was measured at room temperature (24 °C). The sciatic nerve was stimulated supramaximally at the sciatic notch. The electrical stimulation was in the form of 0.05 ms rectangular waves and its frequency was 10 Hz. We recorded and averaged 64 responses, and measured the latency of the CMAP. The CMAPs of the control group were measured before the first stage (n=6), just before the second stage in the adhesion group (n=9) and during the third stage in all three groups The latency was used to estimate the electrophysiological function because it is used in the context of entrapment neuropathy in clinical conditions.
1.1.2 Histological examination
Immediately after being killed by an overdose of sodium pentobarbital (300 mg/kg), the rabbits were perfused with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3). The sciatic nerve was excised with the surrounding tissues, and a cross-section (at the centre of the thigh) was prepared using Masson's trichrome stain (n=3 in each group).
1.1.3 Biomechanical examination
The sciatic nerves of a separate group of rabbits were transected at knee level and ligated with 1/0 silk. The ligated sciatic nerve was attached to a pull scale (Imada Co. Ltd, Japan), which stretched it at 45° to the neural bed at a speed of 1 cm/s in order to measure the tensile strength that avulsed the sciatic nerve from the neural bed (n=5 in each group).
1.1.4 Statistical analysis
All data are expressed in the form mean±s.d. Fisher's protected least significant difference tests were used for statistical analysis of the results. All differences were considered significant at the P<0.05 level.
1.2 Experiment 2
Crj:CD rats weighing 500 g were anaesthetised by intraperitoneal injection of sodium pentobarbital. The left sciatic nerve was exposed, and HA labelled with fluorescence albumin was poured onto the nerve (n=1). The HA was flushed out with physiological saline 5 min later. Then, the rat was killed by an overdose of sodium pentobarbital, and the sciatic nerve and neural bed (biceps femoris muscle) were extracted. The specimen was frozen in liquid nitrogen, cut into thin cross-sections (7 μm) and examined under a confocal laser microscope (Leica TSC4D).
2. Results
2.1 Experiment 1
2.1.1 Electrophysiological evaluation
The latency of the control group was 1.68±0.07 ms. The latency of the adhesion group was 2.32±0.12 ms. The latencies of the neurolysis group, the HA group and the steroid group were 2.14±0.20, 1.92±0.11 and 1.91±0.15 ms, respectively (Fig. 2) . Thus, the latency of the adhesion group was significantly longer than that of the other groups. We conclude that the neurolysis procedure significantly improved electrophysiological function to the adherent nerve because the neurolysis group was significantly better than the adhesion group. The latencies of the HA and steroid groups were much shorter than that of the neurolysis group but longer than that of the control group. There was no significant difference between the HA and steroid groups.
Fig. 2The results of the electrophysiological study. The latency of the adhesion group was significantly longer than that of the other groups. The latencies of the HA and steroid groups were significantly shorter than that of the neurolysis group but longer than that of the control group. There was no significant difference between the HA and steroid groups.
The tissues, coloured blue with Masson's trichrome, showed fibrous scarring wrapped around the nerve and bridging the nerve and the neural bed. The muscle had already healed from the burn injury caused by the bipolar coagulation in the first stage. The extraneural and intraneural scar tissue in the neurolysis group was remarkable (Fig. 3(A)) . Scar tissue in the neurolysis group was thicker and more voluminous than in the HA group (Fig. 3(B)) or the steroid group (Fig. 3(C)). While the intraneural fibrosis was hardly seen in the HA and steroid groups, the extraneural scarring in the HA group was thinner than that in the steroid group.
Fig. 3Cross-sections through the sciatic nerve and neural bed. The neural bed can be seen at the bottom (Masson's trichrome staining). (A) Neurolysis group: there is fibrous bridging between the nerve and the neural bed; there is no room between the fascicule and the neural bed; and the lower half of the nerve shows intraneural fibrosis. (B) HA group: there is little fibrous bridging between the nerve and the neural bed; and the nerve shows no intraneural fibrosis. (C) Steroid group: there is a little fibrous bridging between the nerve and the neural bed; and the nerve shows no intraneural fibrosis.
The tensile strengths required to remove the sciatic nerve from the neural bed were measured using the pull scale. The tensile strength of the neurolysis group was 6.89±1.73 N, that of the HA group was 3.30±0.57 N and that of the steroid group was 4.88±2.26 N (Fig. 4) . While the tensile strength of the HA group was significantly less than that of the neurolysis group, there was no significant difference between the steroid group and the neurolysis group (P=0.053) or between the HA group and the steroid group (P=0.131).
Fig. 4The results of the biomechanical study. The tensile strength of the HA group was significantly less than that of the neurolysis group. There was no significant difference between the steroid group and the neurolysis group.
Fluorescence staining of the HA was observed in the epineurium and the surface of the muscle (Fig. 5) . We concluded that the HA was completely flushed out and taken into the surrounding tissues because there was no remnant HA in the open space.
Fig. 5The results of experiment 2. (A) A cross-section through the rat sciatic nerve and neural bed, which has been infiltrated for 5 min with HA. The field is photomicrographed under visible rays without additional staining. The bar indicates 100 μm. The enlarged square is the same as that shown in (B). (B) The enlarged square in (A) was observed under the laser-beam microscope. Fluorescence staining of HA (green) is observed in the epineurium and the surface of the neural bed.
When the surgical field of the neurolysis was coated with HA from the beginning of the surgery, adhesion between the nerve and the neural bed was effectively prevented in our model. Foetal wounds heal without scarring, possibly in part because of high levels of HA.
Exogenous HA applied to adult hamster cheek-pouch wounds improves microcirculatory perfusion at the site of tissue repair and accelerates wound closure.
Therefore, maintaining elevated levels of HA in adult wounds may decrease or eliminate scarring. Burns et al. reported effectively inhibiting serosal tissue damage and preventing surgical adhesions by precoating tissues with HA using a rat caecal abrasion model.
Almost immediately after injury, exudate forms a thin fibrous membrane on the wound site. Native tissue plasminogen activator lyses the fibrous adhesion before it can become permanent. Injured tissues reduce plasminogen-activator activity, thus allowing persistence of the fibrous adhesion.
HA inhibits the reduction of the plasminogen-activator activity. The concept of precoating with HA is the same as ours, because the sciatic nerve and the surrounding tissues were immersed in HA from the beginning to the end of the operation. The tissues injured by incision, separation and strip procedures were protected by HA coating in the earliest stages. HA might prevent the stimulation of the inflammation circuit in the beginning phases. Grainger et al. reported that HA infiltration at the end of surgery did not prevent adhesion.
Hence, HA must be present in the early phase of surgery. When the deep tissue was exposed, it was immediately coated with HA because the surgical field was coated with HA at each step.
In experiment 2, uptake of HA to the surfaces of the surrounding tissues occurred within 5 min. This shows that HA protects the surrounding tissue in the early phase of the injury caused by surgical invasion. Since we felt that early uptake of HA was important to protect the tissues, we used low-molecular-weight HA. High-molecular-weight HA (high HA), 230×104, is more viscous than the low-molecular-weight HA (low HA) we used in this study (84×104). Because of its high viscosity, high HA remained longer and had a more sustained effect than low HA. However, intraarticular injection experiments have revealed that high HA is less distributed in the synovial-lining layers than low HA.
and causes dehydration following the adhesion. Therefore, we used low HA in this study and expect that it acted not as a barrier but as a pharmacological inhibitor of the migration of inflammatory cells. HA also promotes nerve regeneration.
Because it decreases scarring and improves fibrin-matrix formation, HA was added sequentially to the regenerating peripheral rat sciatic nerve via an injectable nerve guide.
Interposition of a pedicle fat flap significantly improves specificity of reinnervation and motor recovery after repair of transected nerves in adjacency in rats.
In our model, there were three steps: producing an adhesion, stripping the adhesion and assessing the outcome. Our model closely resembled the real clinical conditions. We selected a period of 6 weeks because the scarring after 4 months is thinner than after 2 months.
Electrophysiological study demonstrated the effectiveness of neurolysis because the neurolysis group showed a better result than the adhesion group. Thus, simple neurolysis has a positive effect in the clinical situation. However, simple neurolysis did not prevent re-adhesion in the affected nerve. The HA and steroid groups thus showed better recoveries than the neurolysis group. Histological examination showed thick scars, especially intraneurally, in the neurolysis group. Postoperative adhesion and extraneural or intraneural scarring may lead to persistent or new peripheral nerve symptoms.
The connection between the nerve and the nerve bed was thinner in the HA group than in the steroid group. These results closely mirrored the biomechanical results. We measured the tensile strength required to avulse the sciatic nerve from the neural bed. This showed how strongly the nerve was fixed to the neural bed. Because of the thin scarring around the nerve, the sciatic nerve of the HA group was avulsed from the neural bed more easily than that of the neurolysis group.
Various materials can prevent adhesion, including ADCON-T/N, which is simple and easy to prepare because it is a biodegradable gel formulation containing the carbohydrate polymer.
The main effect of ADCON-T/N is to establish boundaries to the cellular migration of fibroblasts. Since the aim of previous studies was to form a barrier between the potentially adherent tissues, their concept is different from ours. We expect HA to exert a pharmacological effect rather than a barrier effect. Using steroid solution as an anti-inflammatory agent in several studies has shown that the steroid reduces adhesion.
In our model, the steroid group had reduced scar formation around the nerve. Therefore, we believe that the anti-inflammatory property of the steroid makes it effective in preventing adhesion. Our study supports clinical use of the steroid at the end of surgery. Because the mechanisms by which HA and the steroid prevent adhesion might be different, a combination therapy of HA (from the beginning to the end of the operation) and the steroid (at the end of surgery) may be effective. Therefore, using HA and the steroid in primary nerve surgery is expected to reduce the need for revisional surgery.
In conclusion, even though re-adhesion occurred around the nerve, simple neurolysis improved nerve function. Steroid infiltration at the end of neurolysis reduced re-adhesion. Coating the surgical field with HA from the beginning to the end of neurolysis reduced re-adhesion around the nerve. Coating with HA was the most effective method of reducing extraneural and intraneural scarring after neurolysis.
Acknowledgements
We thank Mr Hiroshi Yugami for assistance with the electrophysiological study, and Ms Carla Curry Inoue for assistance in presenting this study. We also thank Toyomi Takahashi DVM, PhD from Seikagaku Corporation for assistance with the experiment. We have no personal or institutional financial interest in the drugs, materials or devices described in this paper.
References
McCall T.D.
Grant G.A.
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Treatment of recurrent peripheral nerve entrapment problems.
Interposition of a pedicle fat flap significantly improves specificity of reinnervation and motor recovery after repair of transected nerves in adjacency in rats.
The latency was used to estimate the electrophysiological function because it is used in the context of entrapment neuropathy in clinical conditions.
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