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Effect of low-energy extracorporeal shock wave on vascular regeneration after spinal cord injury and the recovery of motor function

Authors Wang L, Jiang Y, Jiang Z, Han L

Received 14 February 2015

Accepted for publication 6 March 2015

Published 31 August 2016 Volume 2016:12 Pages 2189—2198

DOI https://doi.org/10.2147/NDT.S82864

Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 3

Editor who approved publication: Professor Wai Kwong Tang



This paper has been Retracted 

Lei Wang, Yuquan Jiang, Zheng Jiang, Lizhang Han

Department of Neurosurgery, Qilu Hospital of Shandong University, Jinan, People’s Republic of China

Background: Latest studies show that low-energy extracorporeal shock wave therapy (ESWT) can upregulate levels of vascular endothelial growth factor (VEGF). VEGF can ease nervous tissue harm after spinal cord injury (SCI). This study aims to explore whether low-energy ESWT can promote expression of VEGF, protect nervous tissue after SCI, and improve motor function.
Methods: Ninety adult female rats were divided into the following groups: Group A (simple laminectomy), Group B (laminectomy and low-energy ESWT), Group C (spinal cord injury), and Group D (spinal cord injury and low-energy ESWT). Impinger was used to cause thoracic spinal cord injury. Low-energy ESWT was applied as treatment after injury three times a week, for 3 weeks. After SCI, the Basso, Beattie, and Bresnahan (BBB) scale was used to evaluate motor function over a period of 42 days at different time points. Hematoxylin and eosin (HE) staining was used to evaluate nerve tissue injury. Neuronal nuclear antigen (NeuN) staining was also used to evaluate loss of neurons. Polymerase chain reaction was used to detect messenger RNA (mRNA) expression of VEGF and its receptor fms-like tyrosine kinase 1 (Flt-1). Immunostaining was used to evaluate VEGF protein expression level in myeloid tissue.
Results: BBB scores of Groups A and B showed no significant result related to dyskinesia. HE and NeuN staining indicated that only using low-energy ESWT could not cause damage of nervous tissue in Group B. Recovery of motor function at 7, 35, and 42 days after SCI in Group D was better than that in Group C (P<0.05). Compared with Group C, number of NeuN-positive cells at 42 days after SCI increased significantly (P<0.05). The mRNA levels of VEGF and Flt-1 and VEGF expression at 7 days after SCI in Group D were significantly higher than those in Group C (P<0.05).
Conclusion: Low-energy ESWT promotes expression of VEGF, decreases secondary damage of nerve tissue, and improves recovery of motor function. It can be regarded as one mode of clinical routine adjunctive therapy for spinal injury.

Keywords: spinal injury, impact wave, VEGF, Flt-1, nerve protection

Introduction

At present, extracorporeal shock wave therapy (ESWT) is widely applied in the clinical treatment of various human diseases. Some studies show that ESWT can increase the expression of vascular endothelial growth factor (VEGF) in human umbilical vein endothelial cells cultured in vitro.1,2 At the same time, in the disease model of chronic myocardial ischemia, myocardial infarct, and peripheral vascular disease, low-energy ESWT can increase the expression of VEGF and VEGF receptor, fms-like tyrosine kinase 1 (Flt-1), in vivo and promote vascular regeneration and functional recovery.13 Therefore, VEGF integrated with Flt-1 and together, they play the role of reconstructing the tissue after vascular injury in chronic myocardial ischemia, myocardial infarct, and peripheral vascular disease. VEGF can stimulate angiogenesis and regulate vascular permeability. Many studies have suggested that VEGF produces a neuroprotective effect to reduce secondary neural tissue damage after spinal cord injury (SCI).4,5 Some studies also show that VEGF can stimulate endothelial cells and neural cells, as well as provide neurotrophy, protection, and neural cell proliferation of auxiliary function. Blocking of the endogenous VEGF signaling pathway can cause the death of cells.4,5 There are some studies on the potential of using VEGF to treat SCI.4,5 After SCI, the expression of endogenous VEGF decreased significantly, with worsening of the pathophysiologic process. The therapeutic method for treating this deterioration might include using transcription factor to increase the expression of VEGF or increasing vascularization to inhibit axon degeneration and apoptosis of neurons.69

Although it has been known that low-energy ESWT can increase the expression of endogenous VEGF by noninvasive means after SCI, the effects of low-energy ESWT on VEGF expression and the recovery of motor function have not been investigated in previous studies.10 In this study, we establish a rat model of SCI and explore the effects of low-energy ESWT on the expression of VEGF and recovery of motor function in SCI rats.

Materials and methods

Animals

The conduct of this study agreed with the principles of and was permitted by the ethics committee of Qilu Hospital of Shandong University, (Jinan, People’s Republic of China), who also approved the animal experiments. We divided 90 adult female Sprague Dawley rats (weight: 250–300 g) into four groups randomly, including Group A: sham operation group (simple laminectomy), Group B: simple shock wave therapy (laminectomy and low-energy ESWT), Group C: SCI group (only the spinal cord injury), and Group D: experimental group (spinal injury and low-energy ESWT). Nine rats of each group were used for evaluating motor function. At the same time, hematoxylin and eosin (HE) staining was used for histology analysis of damaged nervous tissue. Three rats in each group were used for evaluating the loss of neurons by neuronal nuclear antigen (NeuN) staining. Four rats were used for real-time polymerase chain reaction (RT-PCR) detection of VEGF and the expression of its receptor Flt-1 at the same time point. Four rats were used for VEGF staining. The rats were kept at normal room temperature with enough food and water before and after the operation.

Establishment of the SCI model and low-energy ESWT treatment

The rats were anesthetized with 1.25% halothane and 30%/70% oxygen and nitrogen gas mixture, with monitoring of rectal temperature to maintain the temperature at 37°C±0.5°C through a heating pad (Fine Science Tools Inc) during the operation. The hair on the back was removed and the spine was washed with disinfectant; a midline incision was made on the skin, the spinal cord was exposed at the T8–T12 vertebral levels for laminectomy, spinal fixation in T8 and T12 was done using a clamp, and then a laminectomy was done from T9–T11 to keep the dural sac complete was done using 10 g heavy-impact rod of the impactor (W.M. Keck Center for Collaborative Neuroscience, NJ, USA). The next step was to close muscle and skin and make the skin in T10 segment vertebral body position using a nylon coil, which could act as a sign of shock wave therapy. After performing the treatment two times per day, the bladder was changed to be empty until they could urinate. The operation group had to be operated consistently with the impact in the group without spinal cord injury. Using the shock wave generator (DUOLITH-SD1, Storz Medical AG; Figure 1A) for low-energy ESWT treatment, shock wave is applied to the two target points near the rat’s T10 tag location. The treatment routine is three times a week for 3 weeks, specifically for the day of SCI and after 2, 4, 7, 9, 11, 14, 16, and 18 days. The same procedure was used to anesthetize rats before every treatment; the shock wave was of 0.1 mJ/mm2 and 4 Hz, striking 200 times per target. According to the product specification, the width of the optimum zone of shock wave was 10 mm, the depth of the probe’s effect was 10 mm (Figure 1B), and the energy was 0.1 mJ/mm2 (positive energy flux density). The process of establishment of the SCI model was based on previous studies.3,5

Figure 1 Process of the low-energy ESWT treatment of rats with SCI.
Notes: A shows that the shock wave probe is in the position between T8 and T10. B shows the best scope of the impact wave.
Abbreviations: ESWT, extracorporeal shock wave therapy; SCI, spinal cord injury.

Analysis of rat behavior

We used the Basso, Beattie, and Bresnahan (BBB) scale to evaluate motor function in rats. This scale, with a range of 0–21 points, can evaluate rat activities, including joint movement, walking, motor coordination, trunk stability, and so on.5 Twenty-one points indicate that the motor function is completely normal. Because some animals only show some amount of motor function recovery, which cannot be shown in the complete BBB score, we also use the BBB subscore (0–13 points) to evaluate rat motor function. The rats were put in the open region and observed for 4 minutes. The rats were evaluated by a trained researcher who was blinded to the grouping. Before the treatment, all the rats were evaluated using the BBB exercise test to make sure that the motor function of rats is normal. The time for evaluating after the operation was 4 hours, 1, 7, 14, 21, 28, 35, and 42 days.

Preparation of tissue and sections

Pentobarbital sodium (100 mg/kg) was injected into the abdominal cavity of rats at 7 and 42 days after SCI. After saline perfusion through the heart, a mixture of 4% paraformaldehyde and 0.1 M phosphate-buffered saline (PBS) was reinjected into the rats. The tissues from the injured parts of the spinal cord were taken and embedded in paraffin overnight with the same fixing solution at the temperature of 4°C. Serial sections were taken at intervals of 250 μm, and the thickness of the sections was 7 μm. The number of sections for each animal was 29. After staining with hematoxylin and eosin to stain the sections, images were obtained using a microscope (BX51, Olympus). No staining was observed in the position of damaged lesions. We also evaluated whether low-energy ESWT has a bad effect on myeloid tissue and also checked tissue injury, including hemorrhage, vacuolation, changes in spindle neurons of the spinal cord, pathological changes of white matter, and so on, in Groups A and B by histological examination. All the evaluations were conducted by experienced staff.

Immunohistochemical analysis

NeuN immunohistochemical analysis was carried out on tissue sections at 42 days after SCI and staining of sections for VEGF at 7 days after SCI. The sections were dewaxed, hydrated, and cleaned for 10 minutes with PBS; after that, a mixture of 0.3% Tween and PBS was used for 10 minutes to rinse the section. The sections were fixed in PBS containing 3% milk and 5% fetal bovine serum (0.01 M) for 2 hours. The tissue sections were treated with mouse anti-NeuN antibody (1:100, MAB377; Merck Millipore) or rabbit anti-VEGF antibody (1:50, sc-152; Santa Cruz Biotechnology) in PBS and incubated overnight at 4°C. After cleaning with PBS, the sections were incubated with goat anti-mouse immunoglobulin G (IgG; Alexa Fluor 488; 1:500, Molecular Probes) or goat anti-rabbit IgG (Alexa Fluor 594) (1:500, Molecular Probes) for 1 hour. Then, we used antifluorescence medium (containing 4′,6-diamidino-2-phenylindole or DAPI, showing cytoplasm) to fix the sections. All the sections were stained simultaneously.

Number of NeuN-positive cells

To explore the loss of neurons in myeloid tissue, we counted the number of NeuN-positive cells. NeuN immunohistochemical staining for spinal cord sections was done at 42 days after SCI. Considering the damaged area as the center, four sections were chosen from the head and tail sides of the vertebrae (1,000 μm and 1,500 μm, respectively) for analysis. The slides were observed under a BX51 microscope, and Photoshop software was used to handle the obtained pictures (version 8.0, Adobe Systems). The number of NeuN-positive cells in each well was manually counted. Their sum is the number of positive cells in the whole section. A number of sections of cells at the same position were compared. The number of NeuN-positive cells of Groups A and B were compared to evaluate the change in the number of neurons of spinal cord tissue that were not damaged after low-energy ESWT treatment.

Quantitative RT-PCR detection

After SCI, at 7 and 21 days, a spinal cord segment (of length about 10 mm) in the SCI region without bacterial infection was obtained. The tissues were homogenized using a POLYTRON unit (Kinematica) and TRIZOL reagent (Invitrogen, USA) was used to extract total RNA from tissues. According to the product manual, RNeasy Mini kit (Qiagen) was used for purification. A large-capacity complementary DNA (cDNA) library kit (Applied Biosystems) was used to synthesize the first strand of cDNA (ABI StepOnePlus, Power SYBR Green PCR MasterMix; Applied Biosystems) for quantitative RT-PCR analysis. Both of them evaluate the messenger RNA (mRNA) expression of VEGF and its receptor Flt-1. Each primer (final concentration of 500 nM) was designed based on GenBank database (VEGF, forward (F): 5′-GAGTTA A ACGA ACGTACTTGCAGA-3′, reverse (R): 5′-TCTAGTTCCCGAAACCCTGA-3′; Flt-1, F: 5′-CAGTTTCCAAGTGGCCAGAG-3′, R: 5′-AG GTCGC GATGAATGCAC-3′; β-actin, F: 5′-CCCGC GAGTACA ACCTTCT-3′, R: 5′-CGTCATCCATG GCGAACT-3′). StepOne software’s comparative Ct method was used to quantify the recurring number of the fluorescence threshold (Ct value). Ct value (Ct[GOI]) of the target gene was standardized using the value of β-actin. The result shows that −ΔCt = −(Ct[GOI] – Ct[β-actin]).

Immune strength in terms of VEGF staining

To evaluate the expression of VEGF protein in myeloid tissue after being damaged, we evaluated the immune strength of VEGF antibody by staining the tissue slices at 7 days after SCI. The photo of the whole cross section was taken using BX51 microscope under ×10 magnification. We chose a position at 1,000 μm and 1,500 μm from the damaged end of each animal and took four sections for analysis. To avoid image error, microscope parameter settings for all the sections were the same. ImageJ software was used to analyze damaged area of the whole section and complete peripheral region. By setting the automatic threshold to obtain section-specific signal threshold, immune strength beyond the threshold is automatically recorded.

Statistical analysis

Repeated measure analysis for variance was used to analyze the motor function difference between groups of 1- to 6-week-old rats. The nonpaired t-test was used to quantitatively evaluate RT-PCR, the NeuN-positive cell count, and VEGF immunostaining intensity. If P<0.05, the differences were considered to be of statistical significance. All the data were analyzed by SPSS 16.0 software.

Results

BBB motor function score

To evaluate the effect of low-energy ESWT on motor function, BBB grade and fraction were used to evaluate the latter at 6 weeks after operation. The result showed that the scores of rats for Groups A and B were the maximum, and there was no impairment of motor function (Figure 2A and B). The recovery of motor function at 7, 35, and 42 days of Group C after SCI was better than that of Group D (P<0.05, Figure 2C). At 42 days, the BBB score in Group C was 14–18 (mean value is 17±1.6), and that for Group D was 12–14 (mean value is 13±0.9). In Group C, the BBB score of one rat was 14 points, and all the remaining five rats had a good walking posture with good coordination of front and hind legs. At 42 days, the main positions of the feet touched and left the ground in parallel. In Group D, four rats could not maintain parallel positions of the feet while walking, with motor coordination of front and hind legs appearing occasionally. The BBB score of Group C at 14, 21, 28, 35, and 42 days after SCI was higher than that of Group D (P<0.01, Figure 2D).

Figure 2 BBB fraction and scores of the rats.
Notes: (A) The BBB scores of Groups A and B. (B) The BBB fractions of Groups A and B. The BBB scores are the maximum for Groups A and B 6 weeks after operation, with no motor dysfunction. (C) The BBB scores of Groups C and D. (D) The BBB fractions of Groups C and D. The BBB score of Group C at 7 days, 35 days, and 42 days after operation is obviously lower than the same for Group D (*P<0.05); the BBB fraction of Group C at 14 days, 21 days, 28 days, 35 days, and 42 days is significantly lower than that for Group D (**P<0.01).
Abbreviations: BBB, Basso, Beattie, and Bresnahan scale; SCI, spinal cord injury.

HE staining of tissue section

Figure 3AD shows that there was no spinal injury in Groups A and B at 7 and 42 days after SCI. The use of only low-energy ESWT did not cause hematomyelia, vacuolated cells, and fusiform neurons change. Figure 3EH shows the form of hemorrhage and vacuolated cells in spinal cord of Groups C and D.

Figure 3 HE staining of myeloid tissue.
Notes: (A and B) Transverse section of spinal cord of rats from Groups A and B. There is no spinal cord tissue injury. (C and D) Enlarged view of the sections, but there is still no visible tissue injury. (E and F) The form of vacuolated cells in the damaged regions of spinal cord in rats of Groups C and D. (G and H) Enlarged sections show hemorrhage, follicular and other tissue injury in SCI. (I) Position of sectioning. All scale bars are 500 μm.
Abbreviations: HE, hematoxylin and eosin; SCI, spinal cord injury.

Number of NeuN-positive cells

To evaluate the loss of neuronal cells in undamaged spinal cord tissue after using low-energy ESWT, we compared the number of NeuN-positive cells of Groups A and B at 42 days after SCI. The result shows that the number of neuronal cells in the two groups are almost the same (Figure 4AL). Use of low-energy ESWT did not influence the neuronal cells in undamaged spinal cord tissue (Figure 4N). To evaluate the neuroprotective effect of low-energy ESWT after SCI, we compared the number of NeuN-positive cells at 42 days in Groups C and D (Figure 5AL). The result shows that in the position of the site of the SCI center (1,000 μm), the number of positive cells in Group D was higher than that in Group C (313 and 53.2 vs 78.3 and 69.9, respectively; P=0.010; Figure 5N).

Figure 4 NeuN immunohistochemical staining of Groups A and B at 42 days after SCI.
Notes: (AF) The number of NeuN-positive cells of the two groups is basically the same. Scale bars are 200 μm. (GL) The enlarged image of sections showing NeuN-positive cells (shown by arrowheads; scale bars are 50 μm). (M) Position of sectioning. (N) The differences between the two groups are not statistically significant.
Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; NeuN, neuronal nuclear antigen; SCI, spinal cord injury.

Figure 5 NeuN immunohistochemical staining analysis of Groups C and D at 42 days after SCI.
Notes: (AF) In the position of the site of the SCI center at 1,000 μm, the number of positive cells in Group D is higher than that of Group C. Scale bars are 200 μm. (GL) The enlarged section shows scattered NeuN-positive cells in Group C (arrowheads). Scale bars are 50 μm. (M) Section position. (N) In the position of the site of the SCI center at 1,000 μm, the number of positive cells in Group D is higher than that of Group C (*P=0.01).
Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; NeuN, neuronal nuclear antigen; SCI, spinal cord injury.

mRNA expression of VEGF and Flt-1

The mRNA expression levels of VEGF and Flt-1 at 7 days after SCI for Group C (P=0.018) were obviously higher than those in Group D (P=0.004; Figure 6). At 21 days, the mRNA levels of Groups C and D were higher than those at 7 days.

Figure 6 The mRNA levels of VEGF (A) and its receptor Flt-1 (B) in rats of Groups C and D at 7 and 21 days after SCI.
Notes: *P<0.05, **P<0.01.
Abbreviations: Flt-1, fms-like tyrosine kinase 1; mRNA, messenger RNA; SCI, spinal cord injury; VEGF, vascular endothelial growth factor.

Immune strength in terms of VEGF staining

We evaluated the protein expression of VEGF at 7 days after SCI and compared the immune strengths of VEGF antibody staining of Groups C and D. The result shows that the frequency of VEGF-positive cells appearing in Group D was higher than that for Group C (Figure 7AL). The position with the most obvious difference in the tissue section was located in the scope at a distance of 1,000 μm away from the cephalad and caudal regions of injury center. Obvious difference exists in the immune strength groups of injury center (P=0.009, Figure 7N).

Figure 7 VEGF immunohistochemical staining analysis.
Notes: (AF) It is easier to find VEGF-positive cells in the injury center of Group D; scale bars are 100 μm. (GL) Enlarged view of VEGF-positive cells; scale bars are 25 μm. (M) Section position. (N) VEGF staining strength in SCI center of Group D is higher than that in Group C (**P=0.009).
Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; SCI, spinal cord injury; VEGF, vascular endothelial growth factor.

For the Flt-1 protein, we also examined, using the immunochemistry assay, the Flt-1 protein and found results with similar changes as with VEGF expression (data not shown).

Discussion

The result of this study preliminarily confirms that low-energy ESWT does not have a bad effect on myeloid tissue and does not damage the motor function of rats. It can obviously increase the expression levels of VEGF protein and mRNA at 7 days after SCI, reduce the loss of neurons, and improve the recovery of motor function. It is the first time that a study has confirmed that low-energy ESWT can promote the neuroprotective effect of VEGF and the recovery of motor function after SCI, which has very important potential application value for clinical SCI treatment.

The effect of the impact wave on tissues and organs is related to pressure. High-energy shock waves can cause microfracture, hematoma formation, and other bad consequences. At the same time, they can also destroy the neurovascular structure of brain and spinal cord. Histological study shows that high- and low-energy shock waves can cause the increase of neurons in spinal cord or brain tissue, improve the hemorrhagic contusion and change in fusiform neurons.1113 The results of this study show that low-energy ESWT does not cause damage to the nervous tissue by counting NeuN-positive cells, which show that it does not exacerbate the death of neurons.12 Moreover, only using low-energy ESWT does not influence the motor function of rats; therefore, low-energy ESWT does not cause the damage of myeloid tissue. It is safe to apply it for the treatment of spinal injury.

The use of impact wave can cause vacuolated cells. The physical force can produce blade load in the local cell membrane, which can lead to various biochemical effects.14,15 This study shows that the impact wave can increase the expression of VEGF and its receptor Flt-1 in tissues and organs; moreover, low-energy ESWT can increase the expression of VEGF and Flt-1 in endothelial cells. In chronic myocardial ischemia, acute myocardial infarction, or peripheral vascular disease models, low-energy ESWT can increase the expression of VEGF in ischemic tissues.16 It can increase the generation of lymph vessels in secondary lymphedema model of rats. At the same time, some studies show that it can speed up the rate of healing of wounds in diabetic mice.17 In the present study, we confirm that low-energy ESWT can significantly strengthen VEGF expression and its receptor in myeloid tissue, which could enhance the biological effect of VEGF on the damaged nerve tissue in the SCI model.

During the development of the nervous system, VEGF is very important for tissue vascularization and nerve cell proliferation.18 In normal situation, the mRNA and protein expression level of VEGF decrease during the acute and subacute periods of SCI (12 hours to 14 days), which causes the loss of endogenous neural tissue protection function and secondary tissue damage; therefore, the increased expression of VEGF and its receptor can recover the local blood supply, improve survival and repair of nerve tissue, and promote the recovery of motor function.1922 A previous study9 has shown that recombinant VEGF expression with adenoviral vector can promote the regeneration of corticospinal tract axons of spinal cord transection in rats. Some studies confirm that the neuroprotective effect is achieved by the antiapoptotic pathway, which can lead to the release of NO, angiogenesis, and antiapoptosis.23 In this study, after being treated with low-energy ESWT, the expression level of VEGF and its receptor increased, neuroprotective effect was strengthened, and the recovery of motor function was better than that in the untreated group.

Impact wave can regulate the expression of VEGF, apart from leading to varied biological effects, such as nonenzymatic NO synthesis, Ras activation, and the expression of matrix metalloproteinase and many chemokines that exert anti-inflammatory effect.24 Park et al25 confirm that ESWT can strengthen endothelial NO synthase activity, and the production of NO increases in cells. At the same time, the enhanced anti-inflammatory effect is also reflected in the decreased expression of NF-κΒ and its dependent gene.26 ESWT can also strengthen the expression of BMP-2 and transforming growth factor β,27,28 so applying low-energy ESWT for treatment after SCI has neuroprotective effect.29 At present, the expression of which cell substance contributes to the VEGF increases in spinal cord is still not clear, and its corresponding specific matrix is needed to be confirmed by further research. Until now, some cells have been discovered with the potential for expressing VEGF and its receptor Flt-1.3032 All of these tissues or cells would provide some clues about the expression of VEGF.

In clinical practice, application of low-energy ESWT to treat SCI to patients’ back is advisable.33 Because of the presence of sclerostin and metal that affect the impact wave, the effect of treatment of diseased region after laminectomy is better than the result of other fixation surgeries.34 The biggest advantage of impact wave treatment is its noninvasive and safe profile, there being no side effect and complication of the procedure. According to patients, repeated treatment can be applied instead of anesthesia, catheter intervention, and taking medication.

In conclusion, low-energy ESWT can strengthen the neuroprotective effect of VEGF after SCI, reduce the secondary injury to nervous tissue, and improve the recovery of motor function of SCI rats. Therefore, we speculate that low-energy ESWT may be applied as one of the routine clinical adjunctive therapies for spinal injury. This study should be followed by further clinical studies.

Disclosure

The authors report no conflicts of interest in this work.


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