Brain-derived neurotrophic factor expression in dorsal root ganglion neurons in response to reanastomosis of the distal stoma after nerve grafting.

Studies have shown that retreatment of the distal stoma after nerve grafting can stimulate nerve regeneration. The present study attempted to verify the effects of reanastomosis of the distal stoma, after nerve grafting, on nerve regeneration by assessing brain-derived neurotrophic factor expression in 2-month-old rats. Results showed that brain-derived neurotrophic factor expression in L2-4 dorsal root ganglia began to increase 3 days after autologous nerve grafting post sciatic nerve injury, peaked at 14 days, decreased at 28 days, and reached similar levels to the sham-surgery group at 56 days. Brain-derived neurotrophic factor expression in L2-4 dorsal root ganglia began to increase 3 days after reanastomosis of the distal stoma, 59 days after autologous nerve grafting post sciatic nerve injury, significantly increased at 63 days, peaked at 70 days, and gradually decreased thereafter, but remained higher compared with the sham-surgery group up to 112 days. The results of this study indicate that reanastomosis of the distal stoma after orthotopic nerve grafting stimulated brain-derived neurotrophic factor expression in L2-4 dorsal root ganglia.

Research Highlights

We hypothesized that reanastomosis of the distal stoma in nerve grafting may stimulate nerve regeneration, and we attempted to verify this hypothesis by assessing brain-derived neurotrophic factor expression in dorsal root ganglia.

The distal stoma was resected and reanastomosed 56 days after autologous nerve grafting, and brain-derived neurotrophic factor expression was secondarily increased.

Reanastomosis of the distal stoma stimulated axons, which regenerated to the distal stoma and activated related neurons to promote axon growth.

This study provides a simple and feasible technique for clinical nerve repair and provides an experimental basis and theoretical evidence for the timing and method for repair of the distal stoma after nerve grafting.

INTRODUCTION

Currently, autologous nerve grafting is the gold standard treatment for long nerve defects[1]. Because of problems associated with nerve regeneration, such as abnormal growth and neuroma formation, the results of long nerve grafting are currently unsatisfactory[2]. Studies have shown that retreatment of the distal stoma after nerve grafting can stimulate nerve regeneration, but the mechanism of regeneration remains unclear[345]. Brain-derived neurotrophic factor is an important neurotrophin involved in nerve regeneration after peripheral nerve injury[67]. The expression of endogenous brain-derived neurotrophic factor was shown to be upregulated in neurons after axotomy[8910111213]. Brain-derived neurotrophic factor promotes nerve regeneration in a number of different ways[1415]. Since nerve injury can induce brain-derived neurotrophic factor upregulation, we hypothesized that reanastomosis of the distal stoma to the grafting nerve may stimulate nerve regeneration. To verify this hypothesis, the present study used a model of orthotopic nerve grafting after sciatic nerve dissection, and reanastomosis of the distal stoma after resecting the distal stoma 56 days after nerve grafting. The expression of brain-derived neurotrophic factor in dorsal root ganglia was detected by reverse transcription-PCR and western blot analysis.

RESULTS

Quantitative analysis of experimental animals

A total of 288 rats were used and randomly assigned to three groups: sham-surgery (the sciatic nerve was exposed alone), sciatic nerve injury (the sciatic nerve was dissected at 15 mm, followed by autologous nerve grafting), and retreatment (the sciatic nerve was treated as the sciatic nerve injury group, but the distal stoma was retreated 56 days later). Eight rats from each group were selected at 7, 14, 28, 42, 56, 59, 63, 70, 84, 96, and 112 days after nerve grafting. All 288 rats were included in the final analyses.

Brain-derived neurotrophic factor mRNA expression in L2-4 dorsal root ganglia

Reverse transcription-PCR results showed that there was minimal brain-derived neurotrophic factor mRNA expression in the sham-surgery group throughout the entire experiment (P > 0.05). Brain-derived neurotrophic factor mRNA expression in the sciatic nerve injury group began to increase 3 days after nerve grafting, peaked at 14 days, decreased at 28 days, and reached similar levels to the sham-surgery group at 56 days. Brain-derived neurotrophic factor mRNA expression in the retreatment group was similar to the sciatic nerve injury group during the first 56 days. After retreatment, brain-derived neurotrophic factor mRNA expression began to increase after 3 days (59th day), significantly increased up to 63 days, peaked at 70 days, and gradually decreased thereafter, but remained significantly higher compared with the sciatic nerve injury group up to 112 days (P < 0.01; Figure 1, Table 1).

Figure 1

Brain-derived neurotrophic factor (BDNF) mRNA expression following retreatment 56 days after autologous nerve grafting as detected by reverse transcription-PCR.

Total RNA was isolated from the dorsal root ganglion in the retreatment group (A), sciatic nerve injury group (B), and sham-surgery group (C). The bands, corresponding to different time points after surgery, were amplified.

I: BDNF mRNA; II: GAPDH mRNA.

Table 1

Changes in brain-derived neurotrophic factor mRNA in dorsal root ganglia at different time points after injury in different groups

Brain-derived neurotrophic factor protein expression in L2-4 dorsal root ganglia

In the sham-surgery group, there was a low level expression of brain-derived neurotrophic factor in the dorsal root ganglion neurons throughout the experiment. However, in the sciatic nerve injury group, brain-derived neurotrophic factor expression began to increase 3 days after nerve grafting, peaked at 14 days, slightly decreased until the 28th day, and significantly decreased up to 56 days similar to levels in the sham-surgery group. In the retreatment group, the change in brain-derived neurotrophic factor protein level was similar to the sciatic nerve injury group for the first 56 days. Three days after the second injury, 59 days after the first injury, the expression of brain-derived neurotrophic factor began to increase again, and was significantly increased at 63 days (7 days after retreatment), and peaked at 70 days (14 days after retreatment). Although there was a slight decrease after this period, there was a significant difference between the sciatic nerve injury and retreatment groups until the 112th day (56 days after retreatment; P < 0.01; Figure 2 and Table 2).

Figure 2

Brain-derived neurotrophic factor (BDNF) protein expression following retreatment 56 days after autologous nerve grafting as detected by western blot.

Equal amounts of protein samples were analyzed in the retreatment group (A), sciatic nerve injury group (B), and sham-surgery group (C). The BDNF (12 kDa) bands, corresponding to different time points after surgery, were amplified. β-actin (42 kDa) immunoblots were used to confirm equal protein loading.

I: BDNF protein; II: β-actin protein.

Table 2

Brain-derived neurotrophic factor protein expression in dorsal root ganglia at different time points after injury in different groups

DISCUSSION

In this study, the change in brain-derived neurotrophic factor expression after reanastomosis was the same in the retreatment group as after the first anastomosis. This indicates that regeneration of the axon passed through the distal stoma. After we cut and reanastomosed the distal stoma, the regenerated axon was cut again. The neurons received the injury signal and presented the same changes in brain-derived neurotrophic factor associated with the first injury.

Peripheral nerve injury can cause neuronal apoptosis, but a previous study indicated that there was little influence on neurons[16]. In another study, a conditional injury to the sciatic nerve was shown to be beneficial to the ascending fiber[17].

Peripheral nerve injury can stimulate neurotrophin release, including brain-derived neurotrophic factor. A second injury to the same nerve cannot facilitate nerve regeneration, but this is not the case with a conditional injury. A conditional injury refers to the fact that another injury after the first injury has occurred, and this second injury is based on the first injury. Conditional injury causes a series of reactive changes in the corresponding motoneuron and promotes the synthesis of proteins associated with growth[18]. These proteins play a role in accelerating axon regeneration after the second injury. Sjöberg et al[17] found that a conditional injury could not only increase the speed of nerve regeneration, but also shorten the initial arrest of nerve regeneration. Traditional theory suggests that another injury should exacerbate the initial nerve injury. However, Kwon et al[16] showed that a second injury only caused 13% motoneuron injury. Ten weeks after facial nerve injury, a second operation can stimulate nerve regeneration and promote associated gene expression such as growth associated protein-43 and α-tubulin. In contrast to a conditional injury, retreatment of the distal stoma is not a second injury. With a conditional injury, motoneurons do not recover from the first injury before the second injury occurs. Thus, the injury is amplified. In contrast, when the distal stoma is retreated, motoneurons have recovered from the first injury. Thus, injury to motoneurons is considerably less compared with a conditional injury. Additionally, in nerve grafting, the grafting nerve and the distal nerve change in shape because of scar tissue around the nerve and the collapse of the endoneurial tube. Thus, we decided to cut and reanastomose the distal stoma in our nerve grafting model.

Clinically, the functional recovery of long nerve defects is poor after surgery[1] as it is difficult to promote and maintain regeneration. According to previous studies concerning nerve regeneration, proteins involved in nerve regeneration, such as brain-derived neurotrophic factor, are highly expressed within 8 weeks after nerve injury[19]. Taken together, available data suggest that nerves can regenerate only in relation to an injury, and that it is possible that only short-term high level expression of proteins associated with growth causes short-term nerve regeneration. Furthermore, additional injuries can also stimulate nerve regeneration. However, the appropriate time to apply a second injury is when the regenerated axon reaches the distal stoma and can cause axonal injury. The speed of nerve regeneration is approximately 1 mm per day[20]. Thus, nerve regeneration could reach the distal stoma 8 weeks after nerve grafting.

Results from the present study demonstrate that reanastomosis of the distal stoma at an appropriate time after sciatic nerve grafting can promote brain-derived neurotrophic factor expression in dorsal root ganglia and stimulate nerve regeneration.

MATERIALS AND METHODS

Design

A randomized controlled animal study.

Time and setting

Experiments were performed at the Key Laboratory for Molecular Enzymology and Engineering, Ministry of Education, Jilin University, China from May 2009 to December 2011.

Materials

A total of 288 male Wistar rats, weighing 250–300 g, aged 2 months old, were provided by the Laboratory Animal Centre of Jilin University (license No. SCXK (Ji) 2009-0003). All animals were housed from birth under standard cycling and housing conditions (temperature: 22 ± 1°C, relative humidity 60 ± 3%, 12-hour light/dark cycle; minimal noise disturbance), and allowed free access to a standard diet of pellets with water, with five and, subsequently, one rat per cage. All experimental protocols were conducted in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China[21].

Methods

Sciatic nerve injury and repair

After anesthesia with 10% chloral hydrate, 3 mL/kg, rats were placed in a prone position on the operating table and stabilized. The right hip was sterilized with iodophor. After an oblique incision was made, just at the lower border of the piriformis, the biceps flexor cruris was separated and the sciatic nerve exposed. In the sham-surgery group, after the sciatic nerve was exposed, the nerve was marked at two points: the lower border of piriformis, and 15 mm distal to the former. The incision was then closed. In the sciatic nerve injury group, the sciatic nerve was cut at the two marked points and anastomosed. In the retreatment group, 56 days after treatment as in the sciatic nerve injury group, the distal stoma was resected and reanastomosed (supplementary Figure 1 online).

Preparation of L2-4 dorsal root ganglia

After anesthesia with 10% chloral hydrate, 3 mL/kg, the rats were placed in a prone position on the table. After laminectomy, the L2-4 dorsal root ganglia were harvested and placed in liquid nitrogen for reverse transcription- PCR and western blot analysis.

Reverse transcription-PCR for brain-derived neurotrophic factor mRNA expression

Total RNA (3 μg) was extracted from dorsal root ganglia and converted to cDNA with superscript reverse transcriptase (Promega, Madison, WI, USA) and nucleotide Oligo dT. Aliquots from the reverse transcription reaction were used for PCR amplification with primer pairs ubiquitously expressing GAPDH as an internal control. The primers were synthesized by Shanghai Sangon Biological Engineering Co., Ltd., Shanghai, China, and were as follows:

Brain-derived neurotrophic factor: forward primer 5’-GCC CAT ATG ACC ATC CTT TTC CTT A-3’, reverse primer 5’-CTA TCT TCC CCT TTT AAT GGT CAG -3’, the length was 25 bp; GAPDH: forward primer 5’-ACC ACA GTC CAT GCC ATC AC-3’, reverse primer 5’-TCC ACC ACC CTG TTG CTG TA- 3’, the length was 20 bp.

The PCR reactions contained 2.0 μL dNTP mixture (Takara, Tokyo, Japan), 2.0 μL forward primer, 1.0 μL dNTP (10 mM), 5.0 μL buffer, 2.0 U Taq DNA polymerase (Toyobo, Osaka, Japan) 1.0 μL of cDNA added to ddH2O to make a final volume of 50 μL.

The PCR reaction was cycled 30 times at 94°C for 5 minutes, 95°C for 30 seconds, 55°C for 30 seconds and 72°C for 1 minute. The reaction products were electrophoretically separated on 1.5% agarose gel and visualized by staining with ethidium bromide. Data were expressed as the mean absorbance ratio of brain-derived neurotrophic factor band to GAPDH band in the same sample. The absorbance was measured at five points for each band using Quantity One software (Bio-Rad, Hercules, CA, USA).

Western blot assay for brain-derived neurotrophic factor protein expression

Approximately 5 mg of dorsal root ganglion tissue was lysed in ice-cold lysis buffer containing 1 mM sodium orthovanadate, 1 mM NaF, and protease inhibitors. Protein concentration was determined by the Bio-Rad assay (Bio-Rad Laboratories, Hercules, CA, USA). For immunoblotting, proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA), followed by incubation with primary antibody (rabbit anti-rat monoclonal antibody; 1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C and secondary antibody (goat anti-rabbit IgG; 1:2 000; PeproTech Inc., Rocky Hill, NJ, USA) for 2 hours at 37°C. Bound antibody was detected using an enhanced chemiluminescence kit (Amersham Biosciences, Buckinghamshire, England), followed by membrane exposure to film (Kodak, Rochester, NY, USA), developing, and fixing. The absorbance of the scanned bands was determined using Image J (National Institutes of Health, Rockville, MA, USA). β-actin was used as an internal reference.

Statistical analysis

SPSS 17.0 software (SPSS, Chicago, IL, USA) was used for statistical analysis. Relative absorbance data of corresponding bands by reverse transcription-PCR and western blot analysis were expressed as mean ± SD. Statistical differences between different groups were analyzed using two-way analysis of variance followed by the least significant difference t-test. P < 0.05 was considered statistically significant.