Keywords
biofilm - neurosurgical procedure - peripheral nerve injuries - rats - sciatic nerve
Palavras-chave
biofilme - procedimento neurocirúrgico - lesões nervosas periféricas - ratos - nervo
ciático
Introduction
The gold standard in repairing neurotmesis has been the autogenous free nerve graft.
However, the surgical tubing technique, which consists of suturing the neural stumps
inside the guide tube, has been studied since 1980,[1] bringing some benefits such as the prevention of neuromas, adequate guidance for
aligning the stumps, inhibition of fibroblast infiltration, and, consequently, reduced
scar tissue formation in the injured site.[2] This ultimately promotes the formation of a new extracellular matrix and, therefore,
successful nerve regeneration.[3]
Several materials are made to serve as guide tubes for the nerve regeneration process,
among which those of biological origin (muscles, blood vessels, tendons) and those
of natural and synthetic origin stand out.[4]
[5] Materials of synthetic origin can have their mechanical, chemical, and structural
properties modified to increase the nerve regeneration through incorporating substances
in its structure or its production in the form of mesh, sponge, and solid or porous
tubes. The nervous conduit must be non-toxic with sufficient strength and flexibility,
but without pressing the nerve, minimally immunogenic and simple to manufacture. In
addition, it must guide the direction of the regenerative nerve, isolate the regenerated
axon from the scar tissue, and protect the regenerated nerve against surrounding compression.[6] Synthetic conduits in the literature can be classified into non-biodegradable and
biodegradable materials.[7]
Although models for crush injury (neuropraxia) already exist and have been described
in the literature,[8] and there is wide availability of artificial conduits,[9] which are initially tested on animals (mostly rats) to be later applied in clinic,[10] there are no standardized protocols which evaluate the behavior of these materials
in experimental nerve repair models of a neurotmesis using the tubing technique.
Therefore, the present study aims to establish a surgical protocol for nerve repair
of a neurotmesis using the sciatic nerve tubing technique in Wistar rats, using a
polyvinyl alcohol conduit and a water-soluble, non-biodegradable polymer as a model.
This model is well-accepted in the biomedical environment and has already been described
in the literature for other therapeutic purposes.[11]
[12]
Material and Methods
The study was performed at the neuromuscular plasticity laboratory of the anatomy
department and the nanostructured biodevices laboratory (BIONANO) of the biochemistry
department at Universidade Federal de Pernambuco (UFPE). The sample consisted of 18
Wistar rats, maintained at a temperature of 23 ± 1°C, subjected to inverted light/dark
cycles (12 hours) with commercial diet (Purina, St. Louis, MO, USA) and water ad libitum.
The animals were randomized using the Random Allocation version 2.0 software) at 40
days of age into 3 groups: a) the control group (CG, n = 6), rats that which did not undergo neurotmesis; b) neurotmesis group (NG, n = 6), rats which underwent a complete lesion in the midpoint of the sciatic nerve
with subsequent suture; c) neurotmesis and biofilm group (NBG, n = 6), rats that underwent injury and had the suture covered with biofilm. The sciatic
nerves were then analyzed in the 6th postoperative week.
Manufacturing and Obtaining Polyvinyl Alcohol Biofilm
Polyvinyl alcohol biofilms were produced by Bionano laboratory using 16% PVA and 2%
alginate solutions in a 3:1 concentration.
The solutions were placed in syringes and subjected to electrospinning for 8 and 5 hours,
respectively. The biofilms were subsequently submitted to the cross-linking process
for 24 hours, and then were immersed in a solution of 98.9% methanol, 1% glutaraldehyde
and hydrochloric acid. Next, they went through 5 deionized water baths, were placed
to dry at 8°C for 3 to 7 days, cut into 10 mm × 10 mm squares, and then sterilized
under ultraviolet light for 30 minutes.
Surgical Procedure for Sciatic Nerve Injury
All animals were intraperitoneally anesthetized at the age of 60 days with a 0.05 mL
and 0.1 mL xylazine hydrochloride (Anasedan) (20 mg Kg−1) and ketamine hydrochloride solution (Dopalen) (100 mg Kg−1) for each 100 g of the animal's body weight, which had been previously measured.
Trichotomy was then performed after sedation on the right gluteal region, and then
the area was cleaned with chlorhexidine antiseptic. The animal was sent to the sterile
location, placed in the anatomical position, and the surgical procedure was started.
A longitudinal incision was made in the skin of the posterosuperior region of the
right paw, starting one centimeter below the greater trochanter of the femur in a
diagonal direction and ending near the popliteal fossa at the level of the hamstring
muscles. The sciatic nerve was exposed after disjoining the superficial gluteal and
biceps femoris muscles. The sciatic nerve was visualized using a LEICA Zoom 2,000
Stereo Microscope, 10.5–45x (Leica Camera AG, Wetzlar, Germany), and a tentacle was
placed under the nerve for its isolation with the adjacent tissues. AU: Please, note
that if this is the name of a company, you must provide its complete name and location
(city, state, and country) between parentheses.
Neurotmesis was performed in the animals of the injury groups with surgical scissors,
5 mm proximal to the division of the 3 main branches of the sciatic nerve (tibial,
common fibular, and sural), followed by direct coaptation of the nerve extremities
with fascicular alignment and monofilament suture 7–0 CATGUT (Ethicon Inc., Raritan,
NJ, USA) in about 3 points of the epineurium.[13] In addition, the suture was covered with polyvinyl alcohol biofilm in the NBG. The
animals in the CG only suffered a longitudinal skin incision, disjunction of the muscles
described above, and visualization of the sciatic nerve. Then, muscle and skin suture
with 4–0 nylon monofilament (Somerville) was performed to promote the same surgical
stress as in the other groups ([Fig. 1]).
Fig. 1 Surgical procedure: (A) Animal in the anatomical position; (B) trichotomy; (C) skin incision; (D) disjunction of the superficial gluteal muscles and biceps femoris; (E) visualization of the sciatic nerve (arrow); (F) isolation of the nerve; (G) neurotmesis; (H) neurorrhaphy (arrow); (I) biofilm positioned (arrow); (J) biofilm involving the nerve and sutured; (K) muscular suture; (L) skin suture.
Surgical Care
After applying the anesthetics, the analgesic tramadol hydrochloride (5% -100 mg/kg
of animal weight, diluted in 0.9% saline - 1:1) was applied subcutaneously, being
reapplied every 12 hours for 3 days. Topical antibiotic therapy (rifamycin SV sodium
- 10 mg/ml; neomycin sulfate + bacitracin - 5 mg/g + 250 IU/g) was performed at the
end of the surgery, enrofloxacin antibiotic (10% - 5 mg/Kg of animal weight) was then
administered for 4 days every 24 hours, and the meloxican antiinflammatory (0.2% -
0.1 mL/Kg of animal weight) was applied subcutaneously for 2 days every 24 hours.
All surgical instruments used in the procedure were previously sterilized.
Macroscopic Analysis of the Surgical Technique and Euthanasia of Animals
The animals had their body weight measured in the 6th postoperative week, and then were anesthetized again with a 0.05 ml and 0.1 ml xylazine
(Anasedan) (20 mg.Kg−1) and ketamine hydrochloride solution (Dopalen) (100 mg.Kg−1) for each 100 g of animal weight. The right sciatic nerve was visualized to have
an evaluation of the performed surgical technique, and a macroscopic analysis was
subsequently performed. The evaluation was performed after the experimental period,
according to the questionnaire developed ([Table 1]). The animals received an intracardiac dosage of 1 mL of potassium chloride (KCl)
while still under the effect of anesthetics for their euthanasia.
Table 1
Questionnaire for macroscopic evaluation of surgical repair
|
1. Was the skin suture well repaired?
|
() Yes
|
() No
|
() Not applicable
|
|
2. Have the skin spots broken?
|
() Yes
|
() No
|
() Not applicable
|
|
2.1. If yes, a new suture was performed
|
() Yes
|
() No
|
() Not applicable
|
|
3. Was the muscle suture well repaired?
|
() Yes
|
() No
|
() Not applicable
|
|
4. Was the suture of the muscle absorbed?
|
() Yes
|
() No
|
() Not applicable
|
|
5. Are the stumps aligned structurally?
|
() Yes
|
() No
|
() Not applicable
|
|
6. Was the biofilm absorbed?
|
() Yes
|
() No
|
() Not applicable
|
|
6.1. If not, was the biofilm in place?
|
() Yes
|
() No
|
() Not applicable
|
|
7. Are there neuromas?
|
() Yes
|
() No
|
() Not applicable
|
Statistical Analysis
A database was built in Microsoft Excel, 2016 version, (Microsoft Corp., Redmond,
WA, USA) using the obtained data, and later analyzed using the IBM SPSS Statistics
software, Version 20.0 (IMB Corp., Armonk, NY, USA). The data were expressed in frequency
for the qualitative evaluation obtained through the sciatic nerve macroscopy evaluation
questionnaire, and then they were expressed as mean and standard deviation, and the
Shapiro-Wilk normality test was performed to evaluate body weight. The analysis of
variance (ANOVA) was subsequently used followed by the Bonferroni test, as the data
were normal. A significance level of 5% was adopted (p < 0.05).
Results
[Table 2] represents the sample characterization before the surgical intervention, in which
a similarity was observed between the groups at the beginning of the study.
Table 2
Characterization of the sample before neurotmesis
|
CG (n = 6)
|
NG (n = 6)
|
NBG (n = 6)
|
p
|
|
Preoperative weight (g)
|
267.00 ± 25.13
|
265.00 ± 12.44
|
265.00 ± 25.38
|
0.983
|
Abbreviations: CG, control group; GN, neurotmesis group; GNB, neurotmesis biofilm
group.
ANOVA was used, followed by the Bonferroni test. The level of significance was 5%.
[Table 3] shows the percentage of responses related to the questionnaire for the descriptive
evaluation of surgical repair. The animals of all groups had their skin and muscle
sutures well repaired; however, skin stitches were broken in two animals in the CG
(33.30%), and a new suture was performed after surgery. There was no absorption of
the muscle suture in any of the groups.
Table 3
Frequency of responses to the descriptive evaluation questionnaire 6 weeks after surgical
repair of a neurotmesis in Wistar rats
|
Questions
|
GC
|
GN
|
GNB
|
|
Y
|
N
|
NA
|
Y
|
N
|
NA
|
Y
|
N
|
NA
|
|
1. Was the skin suture well repaired?
|
100%
|
0%
|
0%
|
100%
|
0%
|
0%
|
100%
|
0%
|
0%
|
|
2. Have the skin spots broken?
|
33.30%
|
66.70%
|
0%
|
0%
|
100%
|
0%
|
0%
|
100%
|
0%
|
|
2.1. If yes, a new suture was performed
|
33.30%
|
0%
|
66.70%
|
0%
|
0%
|
100%
|
0%
|
0%
|
100%
|
|
3. Was the muscle suture well repaired?
|
100%
|
0%
|
0%
|
100%
|
0%
|
0%
|
100%
|
0%
|
0%
|
|
4. Was the suture of the muscle absorbed?
|
0%
|
100%
|
0%
|
0%
|
100%
|
0%
|
0%
|
100%
|
0%
|
|
5. Are the stumps aligned structurally?
|
100%
|
0%
|
0%
|
83.30%
|
16.70%
|
0%
|
100%
|
0%
|
0%
|
|
6. Was the biofilm absorbed?
|
0%
|
0%
|
100%
|
0%
|
0%
|
100%
|
0%
|
100%
|
0%
|
|
6.1. If not, was the biofilm in place?
|
0%
|
0%
|
100%
|
0%
|
0%
|
100%
|
100%
|
0%
|
0%
|
|
7. Are these neuromas?
|
0%
|
100%
|
0%
|
33.30%
|
66.70%
|
0%
|
0%
|
100%
|
0%
|
Abbreviations: CG, control group; GN, neurotmesis group; GNB, neurotmesis biofilm
group; N, No; NA, not applicable; Y, yes.
Although the biofilm was not absorbed in the NBG, all the stumps were structurally
aligned 6 weeks after surgery, and there were no neuromas. However, 16.70% of the
animals in the NG had misalignment of the stumps, and 33.30% had neuromas.
Discussion
As neurotmesis is the most severe traumatic injury that can affect the peripheral
nervous system and requires surgical treatment,[14] the present study experimentally evaluated the repair and surgical recovery of a
neurotmesis submitted to the application of a PVA biofilm.
The skin suture rupture in two animals in the CG may have been a consequence of the
animals moving freely, increasing the tissue reaction induced by the suture itself
with softening of the surrounding tissues, delaying the onset of fibroplasia.[15] Despite this, the surgical technique was effective, since perfect asepsis and respect
for the force lines of the tissue are necessary in order for the suture to effectively
heal with natural approach of the edges and without exaggerated tension; this prevented
infection, ischemia, or scarring necrosis,[16] and all animals in the study had good suture repair of both the skin and the muscle.
However, the muscle suture thread was not absorbed in all animals, as nylon monofilament
(Somerville) is a non-absorbable synthetic and inorganic thread which loses 30% of
its original tensile strength in 2 years. It was chosen for this for being a non-reactive
tissue and for its ability to stretch even with its permanence in the body, reducing
the risk of infection and tissue stiffness.[15]
Due to the results observed in the structural alignment of the nerve stumps, the biofilms
used promoted nerve stability after injury, while there was no alignment in 16.70%
of the animals in the NG. This corroborates a recent study which stated that the suture
technique by itself does not guarantee mechanical stability, requiring additional
strategies for better repair, such as biofilm or those already used in research of
suture associated with glue or fibrin conduit.[17]
On the other hand, the biofilms were not absorbed after 6 weeks of surgery to repair
neurotmesis. This is similar to previous findings with the use of a nervous conduit
composed of PVA in the injury of the sciatic nerve, demonstrating the presence of
the material 12 weeks after injury, being surrounded by fibrin capsule and connective
tissue, the result of its biodegradation.[18]
The presence of neuromas in NG is possibly related to a disorganized or incomplete
regeneration of a nerve after its injury, due to the deposition of scar tissue by
fibroblasts.[19]
[20] On the other hand, the absence of this structure in NBG reiterates some of the main
benefits of the tubulization technique in nerve repair, which are the proper orientation
of the axonal stumps toward the distal stump or target tissues, prevention of neuromas,
diffusion of neurotrophic factors released by the damaged nerve, accumulation of extracellular
matrix components, and inhibition of fibroblast infiltration, reducing the formation
of scar tissue at the injury site.[2]
Conclusion
The present study was able to establish a surgical protocol for nerve repair of a
neurotmesis using the sciatic nerve tubing technique in Wistar rats, using a polyvinyl
alcohol conduit and a water-soluble, non-biodegradable polymer as a model. Allied
to this, as the structural alignment and the absence of neuroma are parameters considered
for faster nerve repair,[21] it is believed that the tubing technique tested in the present study proved to be
effective in the sciatic nerve repair process from a macroscopic point of view.
Therefore, following the guidelines of this protocol, we propose that new studies
are carried out to assess the histological and functional repair of the sciatic nerve;
these studies should also include a group that has a distance between the stumps in
order to evaluate axonal growth within the biofilm.
