Low-intensity pulsed ultrasound treatment improved the rate of autograft peripheral nerve regeneration in rat methods
Aim. Evidence-backed execution summary for Low-intensity pulsed ultrasound treatment improved the rate of autograft peripheral nerve regeneration in rat methods from Low-intensity pulsed ultrasound treatment improved the rate of autograft peripheral nerve regeneration in rat.
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rat
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Electrophysiological evaluation
reagent used in the protocol.
- Use
- At the end of 12 weeks after surgery, under anesthesia, the both rat sciatic nerves and posterior gastrocnemius muscle were carefully re-exposed and dissected from surrounding tissues. Stimulating needle electrodes connected to an electrical stimulator (Keypoint; Medtronic company, Denmark) were placed on the proxim...
Evaluation of growth axons
reagent used in the protocol.
- Use
- After the animals were sacrificed, the autograft sciatic nerve was harvested. In order to ensure the integrity of the autograft nerve, normal nerves near the proximal and distal suture points should be separated. Then nerves were frozen sliced longitudinally into 7-µm-thick samples. These samples were incubated...
Western blotting detection for NF-κB p65 expression of gastrocnemius muscle
reagent used in the protocol.
- Use
- Muscle tissues were collected and lysed with RIPA buffer and protease inhibitor. Tissue lysates (10 µg) were separated on a 10% SDS-PAGE gel and transferred to PVDF membranes and blocked with 5% nonfat milk (dissolved in 0.5%TBST) for 1 h at room temperature and then incubated overnight at 4 &...
Evaluation of the axons growth
Ultra-thin sections taken from four groups autograft sciatic nerve at 12-week postoperation observed under electron microscope revealed that the regenerated axons were observed in all groups. The thickness and diameter of axons in low-dose group were larger than those of other groups. Except that the first and the f...
- Use
- Ultra-thin sections taken from four groups autograft sciatic nerve at 12-week postoperation observed under electron microscope revealed that the regenerated axons were observed in all groups. The thickness and diameter of axons in low-dose group were larger than those of other groups. Except that the first and the f...
LIPUS treatment project
On the third day after surgery, the LIPUS treatment with the different doses was started to stimulate the sciatic nerves with an ultrasonic therapeutic apparatus (designed and manufactured by medical ultrasound engineering institute of Chongqing medical university, China). Ultrasonic probe diameter: 2.5 cm, ac...
- Use
- On the third day after surgery, the LIPUS treatment with the different doses was started to stimulate the sciatic nerves with an ultrasonic therapeutic apparatus (designed and manufactured by medical ultrasound engineering institute of Chongqing medical university, China). Ultrasonic probe diameter: 2.5 cm, ac...
Functional analysis
Functional analysis was performed with the Catwalk automated gait analysis system (Cat Walk XT 10.5, Nolddus technology, Netherlands). The apparatus captures footprints through footprints light refraction technology. When a rat traverses the glass floor from one side to the other, high-speed camera under the walkway...
- Use
- Functional analysis was performed with the Catwalk automated gait analysis system (Cat Walk XT 10.5, Nolddus technology, Netherlands). The apparatus captures footprints through footprints light refraction technology. When a rat traverses the glass floor from one side to the other, high-speed camera under the walkway...
Muscle perfusion evaluation
The perfusion of triceps surae was evaluated by gray scale CEUS. CEUS was performed by using the M9 ultrasonic diagnosis instrument (Mindray Medical International Ltd) with an L12-5 transducer (5-12 MHz). The ultrasonography contrast agent was SonoVue ® (0.025 ml.kg-1; Bracco, Milan, Italy) an...
- Use
- The perfusion of triceps surae was evaluated by gray scale CEUS. CEUS was performed by using the M9 ultrasonic diagnosis instrument (Mindray Medical International Ltd) with an L12-5 transducer (5-12 MHz). The ultrasonography contrast agent was SonoVue ® (0.025 ml.kg-1; Bracco, Milan, Italy) an...
Electrophysiological evaluation
At the end of 12 weeks after surgery, under anesthesia, the both rat sciatic nerves and posterior gastrocnemius muscle were carefully re-exposed and dissected from surrounding tissues. Stimulating needle electrodes connected to an electrical stimulator (Keypoint; Medtronic company, Denmark) were placed on the proxim...
- Use
- At the end of 12 weeks after surgery, under anesthesia, the both rat sciatic nerves and posterior gastrocnemius muscle were carefully re-exposed and dissected from surrounding tissues. Stimulating needle electrodes connected to an electrical stimulator (Keypoint; Medtronic company, Denmark) were placed on the proxim...
Evaluation of growth axons
After the animals were sacrificed, the autograft sciatic nerve was harvested. In order to ensure the integrity of the autograft nerve, normal nerves near the proximal and distal suture points should be separated. Then nerves were frozen sliced longitudinally into 7-µm-thick samples. These samples were incubated...
- Use
- After the animals were sacrificed, the autograft sciatic nerve was harvested. In order to ensure the integrity of the autograft nerve, normal nerves near the proximal and distal suture points should be separated. Then nerves were frozen sliced longitudinally into 7-µm-thick samples. These samples were incubated...
Evaluation of growth axons
A 1.5-mm segment of the midpoint of the autograft nerve was dissected. The samples were fixed in 2.5% glutaraldehyde for 6 hours, washed three times with PBS, fixed in 1% osmic acid for 2 hours, washed three times with PBS, dehydrated, and plastic embedded. Transverse sections of semi-thin (0.7...
- Use
- A 1.5-mm segment of the midpoint of the autograft nerve was dissected. The samples were fixed in 2.5% glutaraldehyde for 6 hours, washed three times with PBS, fixed in 1% osmic acid for 2 hours, washed three times with PBS, dehydrated, and plastic embedded. Transverse sections of semi-thin (0.7...
Additional Information
How to cite this article: Jiang, W. et al. Low-intensity pulsed ultrasound treatment improved the rate of autograft peripheral nerve regeneration in rat. Sci. Rep. 6, 22773; doi: 10.1038/srep22773 (2016).
- Use
- How to cite this article: Jiang, W. et al. Low-intensity pulsed ultrasound treatment improved the rate of autograft peripheral nerve regeneration in rat. Sci. Rep. 6, 22773; doi: 10.1038/srep22773 (2016).
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- All variables were presented as mean ± standard variation. The data were analyzed by SPSS 17.0 (SPSS Inc., Chicago, IL). Statistical comparison between the groups was conducted using one-way analysis of variance. A Student-Newman-Kewls (SNK) post-test was performed to compare all pairs of groups. Co...
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Electrophysiological evaluation
At the end of 3-monthsurgery, the recovery indexes of the low-dose and mid-dose treatment groups were significantly increased than the control group ( P = 0.000), and the low-dose group was dramatic obvious. There was no significant difference between the high-dose group and the control group ( P = 0.238). ( ).
Evaluation of the axons growth
The immunopositivity of NF200 proteins was used as markers of axons in the regenerating nerves at 8 weeks and 12 weeks. At 8 weeks, the lengths of the axon growth in three treatment groups were longer than the control group. The lengths of the axon growth of the low-dose group were longer than those of the other two treatment groups. The lengths of the mid-dose group were longer than those of the high-dose group. ( ). At the end of 12 weeks, there was no difference among all four groups.
Evaluation of the axons growth
Midpoint toluidine blue staining of the autograft nerves showed that every group had characteristic appearance of regenerated nerves. Low-dose group had slightly larger, more thickly myelinated axons than the other three groups. The control group had smaller and thinner myelinated axons than the mid-dose group and high-dose group. The regenerated myelinated nerve fiber densities of each group were: control group 12910.8 ± 1764.1395 (n/mm 2 ), low-dose group 19725.6 ± 913.9044 (n/mm 2 ), mid-dose group 16089 ± 1154.3745 (n/mm 2 ) and high-dose group 14172.4 ± 813.7843 (n/mm 2 ). Except that the first and the fourth groups had no significant difference ( P = 0.121), the other groups had significant difference between each other ( P = 0.000). ( ).
Evaluation of the axons growth
Ultra-thin sections taken from four groups autograft sciatic nerve at 12-week postoperation observed under electron microscope revealed that the regenerated axons were observed in all groups. The thickness and diameter of axons in low-dose group were larger than those of other groups. Except that the first and the fourth group had no significant difference ( P = 0.728) ( P = 0.680), other groups had significant difference between each other ( P = 0.000) ( P = 0.001). ( ).
The wet weight gastrocnemius muscles
In this study, gastrocnemius muscles in operation side atrophied seriously. The wet weight recovery ratio of gastrocnemius muscle of each group gradually increased from 2 weeks to 12 weeks after operation. Except for the ratio between the control group and the high-dose group ( P = 0.725), the ratio of other groups had significant difference between each other, ( P = 0.032) ( P = 0.000) ( P = 0.000). The low-dose group increased significantly. ( ).
The wet weight gastrocnemius muscles
Masson trichrome of the cross-sectional gastrocnemius muscle showed that fibers in the low-dose group arranged regularly while control group and high-dose group arranged irregularly. And the area of fibers in low-dose group was the maximum ( P = 0.000). ( ).
The blood perfusion of the gastrocnemius muscles
Through the time-intensity curve, the peak intensity (PI) and the area under the time-intensity curve (AUC) of each group at different time points were obtained. The PI of the control group was lower than other three groups at all time points. The PI of the low-dose group was higher than other three groups and the PI of high-dose group was higher than that of the control group from 4 weeks after operation ( P = 0.000). The AUC of low-dose group were higher than other groups from 4 weeks after operation ( P = 0.002) ( P = 0.000) ( P = 0.000) and the AUC of high-dose group had no significant difference with that of the control group at the end of 12 weeks ( P = 0.077). The PI and AUC of the mid-dose group were in the middle level among four groups all the time. ( ).
Expression of NF-κB p65 protein
Western blotting (WB) was carried out. The expression of of NF-κB p65 protein was no significant difference between control group and high-dose group ( P = 0.661), low-dose group and mid-dose group ( P = 0.388) at 2 weeks after operation. Expression of NF-κB p65 protein in low-dose group was lower than other groups from 4 weeks after operation ( P = 0.000). Protein expression of high-dose group was unstable and was no significant difference with control group at the end of 12 weeks ( P = 0.122). Expression of NF-κB p65 in mid-dose group was higher than in low-dose group and lower than in control group ( P = 0.000). ( ).
Measurement outputs
What raw and processed outputs should exist?
Western blotting (WB) was carried out. The expression of of NF-κB p65 protein was no significant difference between control group and high-dose group ( P = 0.6...
- Raw artifact
- Membrane or gel image with visible bands for target and control proteins
- Processed artifact
- Band quantification and normalized densitometry values
- Reported as
- Relative expression values or fold-change comparisons across groups
At the end of 12 weeks after surgery, in all four groups (5 rats each group, n = 20), the PI and AUC were linearly positive correlated with recovery index of CMAP cu...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Functional analysis was performed with the Catwalk automated gait analysis system (Cat Walk XT 10.5, Nolddus technology, Netherlands). The apparatus captures footprints through...
- Raw artifact
- Per-run gait capture with paw placement, timing, and stride features for each animal
- Processed artifact
- Cleaned gait metrics table and recovery trend summary across timepoints
- Reported as
- Group comparisons of gait indices, stride metrics, or recovery curves
The perfusion of triceps surae was evaluated by gray scale CEUS. CEUS was performed by using the M9 ultrasonic diagnosis instrument (Mindray Medical International Ltd) with an L...
- Raw artifact
- Field or section images captured from matched samples
- Processed artifact
- Selected representative panels with quantified intensity, counts, or area measurements
- Reported as
- Per-group imaging summaries with representative figures and quantified endpoints
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect the raw assay or blot output and retain identifiers for each sample and experimental group.
inferred from protocolPreprocessing / cleaning
At the end of 3-monthsurgery, the recovery indexes of the low-dose and mid-dose treatment groups were significantly increased than the control group ( P = 0.000), and the low-dose group was dramatic obvious.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Western blotting (WB) was carried out. The expression of of NF-κB p65 protein was no significant difference between control group and high-dose group ( P = 0.6...; At the end of 12 weeks after surgery, in all four groups (5 rats each group, n = 20), the PI and AUC were linearly positive correlated with recovery index of CMAP cu...; Functional analysis was performed with the Catwalk automated gait analysis system (Cat Walk XT 10.5, Nolddus technology, Netherlands). The apparatus captures footprints through...; The perfusion of triceps surae was evaluated by gray scale CEUS. CEUS was performed by using the M9 ultrasonic diagnosis instrument (Mindray Medical International Ltd) with an L....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
At the end of 3-monthsurgery, the recovery indexes of the low-dose and mid-dose treatment groups were significantly increased than the control group ( P = 0.000), a...; Midpoint toluidine blue staining of the autograft nerves showed that every group had characteristic appearance of regenerated nerves. Low-dose group had slightly larger, more th...; Ultra-thin sections taken from four groups autograft sciatic nerve at 12-week postoperation observed under electron microscope revealed that the regenerated axons were observed...; In this study, gastrocnemius muscles in operation side atrophied seriously. The wet weight recovery ratio of gastrocnemius muscle of each group gradually increased from 2 weeks...
from paperReporting output
Report representative outputs alongside summary comparisons for Western blotting (WB) was carried out. The expression of of NF-κB p65 protein was no significant difference between control group and high-dose group ( P = 0.6..., At the end of 12 weeks after surgery, in all four groups (5 rats each group, n = 20), the PI and AUC were linearly positive correlated with recovery index of CMAP cu..., Functional analysis was performed with the Catwalk automated gait analysis system (Cat Walk XT 10.5, Nolddus technology, Netherlands). The apparatus captures footprints through..., The perfusion of triceps surae was evaluated by gray scale CEUS. CEUS was performed by using the M9 ultrasonic diagnosis instrument (Mindray Medical International Ltd) with an L....
inferred from protocolStructured statistical methods
At the end of 3-monthsurgery, the recovery indexes of the low-dose and mid-dose treatment groups were significantly increased than the control group ( P = 0.000), a...; Midpoint toluidine blue staining of the autograft nerves showed that every group had characteristic appearance of regenerated nerves. Low-dose group had slightly larger, more th...; Ultra-thin sections taken from four groups autograft sciatic nerve at 12-week postoperation observed under electron microscope revealed that the regenerated axons were observed...; In this study, gastrocnemius muscles in operation side atrophied seriously. The wet weight recovery ratio of gastrocnemius muscle of each group gradually increased from 2 weeks...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
At the end of 3-monthsurgery, the recovery indexes of the low-dose and mid-dose treatment groups were significantly increased than the control group ( P = 0.000), and the low-dose group was dramatic obvious. There was no significant difference between the high-dose group and the control group ( P = 0.238). ( ).
The immunopositivity of NF200 proteins was used as markers of axons in the regenerating nerves at 8 weeks and 12 weeks. At 8 weeks, the lengths of the axon growth in three treatment groups were longer than the control group. The lengths of the axon growth of the low-dose group were longer than those of the other two treatment groups. The lengths of the mid-dose group were longer than those of the high-dose group. ( ). At the end of 12 weeks, there was no difference among all four groups.
Midpoint toluidine blue staining of the autograft nerves showed that every group had characteristic appearance of regenerated nerves. Low-dose group had slightly larger, more thickly myelinated axons than the other three groups. The control group had smaller and thinner myelinated axons than the mid-dose group and high-dose group. The regenerated myelinated nerve fiber densities of each group were: control group 12910.8 ± 1764.1395 (n/mm 2 ), low-dose group 19725.6 ± 913.9044 (n/mm 2 ), mid-dose group 16089 ± 1154.3745 (n/mm 2 ) and high-dose group 14172.4 ± 813.7843 (n/mm 2 ). Except that the first and the fourth groups had no significant difference ( P = 0.121), the other groups had significant difference between each other ( P = 0.000). ( ).
Ultra-thin sections taken from four groups autograft sciatic nerve at 12-week postoperation observed under electron microscope revealed that the regenerated axons were observed in all groups. The thickness and diameter of axons in low-dose group were larger than those of other groups. Except that the first and the fourth group had no significant difference ( P = 0.728) ( P = 0.680), other groups had significant difference between each other ( P = 0.000) ( P = 0.001). ( ).
In this study, gastrocnemius muscles in operation side atrophied seriously. The wet weight recovery ratio of gastrocnemius muscle of each group gradually increased from 2 weeks to 12 weeks after operation. Except for the ratio between the control group and the high-dose group ( P = 0.725), the ratio of other groups had significant difference between each other, ( P = 0.032) ( P = 0.000) ( P = 0.000). The low-dose group increased significantly. ( ).
Masson trichrome of the cross-sectional gastrocnemius muscle showed that fibers in the low-dose group arranged regularly while control group and high-dose group arranged irregularly. And the area of fibers in low-dose group was the maximum ( P = 0.000). ( ).
Through the time-intensity curve, the peak intensity (PI) and the area under the time-intensity curve (AUC) of each group at different time points were obtained. The PI of the control group was lower than other three groups at all time points. The PI of the low-dose group was higher than other three groups and the PI of high-dose group was higher than that of the control group from 4 weeks after operation ( P = 0.000). The AUC of low-dose group were higher than other groups from 4 weeks after operation ( P = 0.002) ( P = 0.000) ( P = 0.000) and the AUC of high-dose group had no significant difference with that of the control group at the end of 12 weeks ( P = 0.077). The PI and AUC of the mid-dose group were in the middle level among four groups all the time. ( ).
Western blotting (WB) was carried out. The expression of of NF-κB p65 protein was no significant difference between control group and high-dose group ( P = 0.661), low-dose group and mid-dose group ( P = 0.388) at 2 weeks after operation. Expression of NF-κB p65 protein in low-dose group was lower than other groups from 4 weeks after operation ( P = 0.000). Protein expression of high-dose group was unstable and was no significant difference with control group at the end of 12 weeks ( P = 0.122). Expression of NF-κB p65 in mid-dose group was higher than in low-dose group and lower than in control group ( P = 0.000). ( ).
Machine-readable layer
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