An anti-inflammatory peptide and brain-derived neurotrophic factor-modified hyaluronan-methylcellulose hydrogel promotes nerve regeneration in rats with spinal cord injury methods
Aim. Evidence-backed execution summary for An anti-inflammatory peptide and brain-derived neurotrophic factor-modified hyaluronan-methylcellulose hydrogel promotes nerve regeneration in rats with spinal cord injury methods from An anti-inflammatory peptide and brain-derived neurotrophic factor-modified hyaluronan-methylcellulose hydrogel promotes nerve regeneration in rats with spinal cord injury.
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rat
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Materials and methods
reagent used in the protocol.
- Use
- Maleimide-KAFAK and maleimide-streptavidin were covalently bonded to the MC by thiol-maleimide click chemistry and biotinylated BDNF was conjugated to MC-streptavidin as previously described. Briefly, MC is carboxylated using 1.5 M sodium hydroxide and an overdose of bromoacetic acid. After purification by dia...
Cell culture and cell proliferation assay
reagent used in the protocol.
- Use
- PC12 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM (Gibco ®; Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (Thermo Fisher Scientific), 0.1 mg/mL streptomycin, and 100 U/mL penicillin. Cultures were incubated a...
KAFAK and BDNF release in vitro
reagent used in the protocol.
- Use
- To explore the profile of KAFAK release by the HAMC hydrogel, we added 25 µL of HAMC-KAFAK/BDNF hydrogels (the concentration of KAFAK: 0.6 µg/mL) to 2 mL sterile PBS. KAFAK release into solution was measured using a fluoraldehyde o-phthalaldehyde assay (Thermo Fisher Scientific), by fluorescence analysis....
KAFAK and BDNF release in vitro
reagent used in the protocol.
- Use
- To explore the profile of BDNF release by HAMC hydrogel, we added 25 µL of HAMC-KAFAK/BDNF hydrogels (the concentration of BDNF: 1 µg/mL) to 2 mL sterile PBS. BDNF release into solution was measured using a rat BDNF ELISA kit (Beijing Solarbio Science & Technology Co., Ltd). Supernatant was collected at 6,...
Quantitative detection of TNF-α, IL-1β, IL-6, and IL-10 by ELISA
reagent used in the protocol.
- Use
- Six rats in each group were killed and segments of spinal cord (10 mm around lesion epicenter) were collected into Eppendorf tubes 7 days after surgery. Lysis buffer was added to dissolve the tissues after washing with PBS and then the tissues were sonicated for 10 seconds to isolate proteins. Subsequently, the tiss...
Tissue preparation and histochemistry
reagent used in the protocol.
- Use
- For immunofluorescence labeling, the tissue sections were rinsed with PBS, treated with blocking solution (0.25% Triton X-100, 5% bovine serum albumin in PBS) for 45 minutes and incubated in blocking solution with the following primary antibodies overnight at 4°C: rabbit anti-βIII-tubulin (1:150, Abcam, Ca...
Swelling properties of HAMC-KAFAK/BDNF hydrogels
Swelling properties were measured in physiologic conditions. Hydrogels were added into 50 mL centrifuge tubes with PBS at 37°C. At different time points, the hydrogels were taken out, and the weight was measured after removal of excess water on the surface. The swelling ratio (SR) = ( W t - W o )/ W o ...
- Use
- Swelling properties were measured in physiologic conditions. Hydrogels were added into 50 mL centrifuge tubes with PBS at 37°C. At different time points, the hydrogels were taken out, and the weight was measured after removal of excess water on the surface. The swelling ratio (SR) = ( W t - W o )/ W o ...
Animals and surgical procedures
All procedures were conducted according to protocols approved by Institutional Animal Care and Use Committee of Logistics College of PAP and all experiments were approved by the Institutional Animal Care and Use Committee of Logistics College of PAP. Adult female Sprague Dawley rats (200-230 g) obtained from t...
- Use
- All procedures were conducted according to protocols approved by Institutional Animal Care and Use Committee of Logistics College of PAP and all experiments were approved by the Institutional Animal Care and Use Committee of Logistics College of PAP. Adult female Sprague Dawley rats (200-230 g) obtained from t...
Functional assessments
All assessments were performed and analyzed by two observers blinded to each group. 1) Hindlimb locomotor function was determined before and after injury and transplantation, using the Basso Beattie Bresnahan (BBB) locomotor rating scale, as described previously. Rats were placed individually in the open field, and...
- Use
- All assessments were performed and analyzed by two observers blinded to each group. 1) Hindlimb locomotor function was determined before and after injury and transplantation, using the Basso Beattie Bresnahan (BBB) locomotor rating scale, as described previously. Rats were placed individually in the open field, and...
Quantitative detection of TNF-α, IL-1β, IL-6, and IL-10 by ELISA
Six rats in each group were killed and segments of spinal cord (10 mm around lesion epicenter) were collected into Eppendorf tubes 7 days after surgery. Lysis buffer was added to dissolve the tissues after washing with PBS and then the tissues were sonicated for 10 seconds to isolate proteins. Subsequently, the tiss...
- Use
- Six rats in each group were killed and segments of spinal cord (10 mm around lesion epicenter) were collected into Eppendorf tubes 7 days after surgery. Lysis buffer was added to dissolve the tissues after washing with PBS and then the tissues were sonicated for 10 seconds to isolate proteins. Subsequently, the tiss...
Tissue preparation and histochemistry
For cavity volume analysis, every twentieth of the tissue sections was selected and stained with H&E. The cavity volume of the spinal cord was calculated using the Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA).
- Use
- For cavity volume analysis, every twentieth of the tissue sections was selected and stained with H&E. The cavity volume of the spinal cord was calculated using the Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA).
Statistical analysis
Statistical analysis was performed using the software package SPSS 19.0 (IBM Corporation, Armonk, NY, USA). All data were presented as the mean ± standard error of the mean. Statistical significance of cell proliferation, BBB scores, and maximal angle of the inclined plane were determined using repeated-measure...
- Use
- Statistical analysis was performed using the software package SPSS 19.0 (IBM Corporation, Armonk, NY, USA). All data were presented as the mean ± standard error of the mean. Statistical significance of cell proliferation, BBB scores, and maximal angle of the inclined plane were determined using repeated-measure...
HAMC-KAFAK/BDNF promotes behavioral outcomes after SCI
To examine the effect of HAMC-KAFAK/BDNF transplantation on the functional recovery of SCI rats, the BBB rating scale was used to measure locomotor functional recovery during the 8 weeks after surgery ( ). All animals performed normal during the locomotor behavior test before injury (21.00±0.00), with a signifi...
- Use
- To examine the effect of HAMC-KAFAK/BDNF transplantation on the functional recovery of SCI rats, the BBB rating scale was used to measure locomotor functional recovery during the 8 weeks after surgery ( ). All animals performed normal during the locomotor behavior test before injury (21.00±0.00), with a signifi...
HAMC-KAFAK/BDNF promotes behavioral outcomes after SCI
In addition to weekly locomotor evaluation, the inclined plane apparatus was used to assess motor function biweekly ( ). There was no significant difference in the maximal angles of inclined plane in the SCI group between 4 and 8 weeks post surgery (4 weeks: 33.62°±4.24° vs 8 weeks: 34.11°±4...
- Use
- In addition to weekly locomotor evaluation, the inclined plane apparatus was used to assess motor function biweekly ( ). There was no significant difference in the maximal angles of inclined plane in the SCI group between 4 and 8 weeks post surgery (4 weeks: 33.62°±4.24° vs 8 weeks: 34.11°±4...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Statistical analysis was performed using the software package SPSS 19.0 (IBM Corporation, Armonk, NY, USA). All data were presented as the mean ± standard error of the mean. Statistical significance of cell proliferation, BBB scores, and maximal angle of the inclined plane were determined using repeated-measure...
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Methods
The anti-inflammatory peptide KAFAKLAARLYRKALARQLGVAA (KAFAK) and brain-derived neurotrophic factor (BDNF)-modified hyaluronan-methylcellulose (HAMC) hydrogel was designed for minimally invasive, localized, and sustained intrathecal protein delivery. The physical and biological characteristics of HAMC-KAFAK/BDNF hydrogel were measured in vitro. SCI model was performed in rats and HAMC-KAFAK/BDNF hydrogel was injected into the injured site of spinal cord. The neuronal regeneration effect was evaluated by inflammatory cytokine levels, behavioral test and histological analysis at 8 weeks post operation.
Materials and methods
Maleimide-KAFAK and maleimide-streptavidin were covalently bonded to the MC by thiol-maleimide click chemistry and biotinylated BDNF was conjugated to MC-streptavidin as previously described. Briefly, MC is carboxylated using 1.5 M sodium hydroxide and an overdose of bromoacetic acid. After purification by dialysis, sulfhydryl groups (100 mg of reactive thiols) were integrated into the carboxylated MC polymer (250 mg) main chain upon reaction with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (750 mg) and 3,3′-dithiobis (propionic dihydrazide, 750 mg), followed by reduction of the disulfide bond with dithiothreitol (1.0 g). Maleimide-labeled streptavidin (mol maleimide: mol streptavidin =9:1, 1.1 mg) and maleimide-KAFAK peptide (59 mg) were then added and mixed with previously synthesized sulfhydryl-MC by stirring in 0.1 M PBS (100 mL) overnight at 4&#...
Materials and methods
HAMC-KAFAK/BDNF hydrogels were prepared by physical blending of HA, MC-KAFAK, MC-streptavidin, and biotinylated-BDNF in artificial cerebrospinal fluid (aCSF) with a final composition of 1.5 wt% HA, 2.5 wt% MC-KAFAK, 0.5 wt% MC-streptavidin, and 0.2 wt% biotinylated BDNF. The HAMC hydrogels were prepared with 1.5 wt% HA and 3 wt% MC for comparison. The above ingredients were sterile-filtered before mixing and sequentially added to the aCSF using a high-speed centrifugal mixer (Eppendorf) for 25 seconds at 3,000 rpm/min and dissolved at 4°C for 12 hours. For in vivo studies, the HAMC-KAFAK/BDNF hydrogel compositions were sterilized by filtration and the preparation process was in sterile conditions. The time interval between preparation and use of the final composite hydrogels was less than 15 minutes, during which time the hydrogels were kept at 4°C ( ).
Swelling properties of HAMC-KAFAK/BDNF hydrogels
Swelling properties were measured in physiologic conditions. Hydrogels were added into 50 mL centrifuge tubes with PBS at 37°C. At different time points, the hydrogels were taken out, and the weight was measured after removal of excess water on the surface. The swelling ratio (SR) = ( W t - W o )/ W o × 100%. W t is the weight of the swollen hydrogel and W o is the initial weight. The SR was calculated at 6, 12, 18, and 24 hours.
Cell culture and cell proliferation assay
PC12 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM (Gibco ®; Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (Thermo Fisher Scientific), 0.1 mg/mL streptomycin, and 100 U/mL penicillin. Cultures were incubated at 37°C, 5% CO 2, and 95% humidity. The cultivated medium was replaced every 2 days. Cells were passaged when confluence reached 80%-90%. HAMC-KAFAK/BDNF treatment was used to evaluate the effect of released BDNF on PC12 cells proliferation. The PC12 cells were seeded at the density of 1 × 10 4 cells/cm 2 into 96-well plates and 100 µL medium per well. Cells were treated with HAMC-KAFAK/BDNF hydrogels, while the same volume of DMEM was added to the control group. The PC12 cells proliferation was evaluated with a CCK-8 cell proliferation kit (Dojindo Molec...
KAFAK and BDNF release in vitro
To explore the profile of KAFAK release by the HAMC hydrogel, we added 25 µL of HAMC-KAFAK/BDNF hydrogels (the concentration of KAFAK: 0.6 µg/mL) to 2 mL sterile PBS. KAFAK release into solution was measured using a fluoraldehyde o-phthalaldehyde assay (Thermo Fisher Scientific), by fluorescence analysis. Fluorescence measurements of KAFAK release were taken at 6, 12, 24, 48, 72, and 96 hours. Three replicates were performed.
KAFAK and BDNF release in vitro
To explore the profile of BDNF release by HAMC hydrogel, we added 25 µL of HAMC-KAFAK/BDNF hydrogels (the concentration of BDNF: 1 µg/mL) to 2 mL sterile PBS. BDNF release into solution was measured using a rat BDNF ELISA kit (Beijing Solarbio Science & Technology Co., Ltd). Supernatant was collected at 6, 12, 24, 48, 72, and 96 hours. Three replicates were performed. Finally, the accumulated release ratio was calculated.
Animals and surgical procedures
All procedures were conducted according to protocols approved by Institutional Animal Care and Use Committee of Logistics College of PAP and all experiments were approved by the Institutional Animal Care and Use Committee of Logistics College of PAP. Adult female Sprague Dawley rats (200-230 g) obtained from the Laboratory Animal Center of the Military Medical Science Academy of China were anesthetized with a nitrous oxide/oxygen mixture (70%/30%) containing 1.4% isoflurane delivered by nose cone. All 72 rats were randomized to 4 groups: sham, SCI, HAMC, and HAMC-KAFAK/BDNF (n=18). A laminectomy was performed at the T10 vertebral level to expose the spinal cord and an aneurysm clip with a force of 25 × g was used for 1 minute to develop an SCI model. A HAMC-KAFAK/BDNF or HAMC hydrogel injection was administered after 5 minutes of SCI. A Hamilton syringe was inserted into th...
Measurement outputs
What raw and processed outputs should exist?
The anti-inflammatory peptide KAFAKLAARLYRKALARQLGVAA (KAFAK) and brain-derived neurotrophic factor (BDNF)-modified hyaluronan-methylcellulose (HAMC) hydrogel was designed for m...
- 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
To explore the profile of KAFAK release by the HAMC hydrogel, we added 25 µL of HAMC-KAFAK/BDNF hydrogels (the concentration of KAFAK: 0.6 µg/mL) to 2 mL sterile PBS....
- 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
To explore the profile of BDNF release by HAMC hydrogel, we added 25 µL of HAMC-KAFAK/BDNF hydrogels (the concentration of BDNF: 1 µg/mL) to 2 mL sterile PBS. BDNF rel...
- 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
All assessments were performed and analyzed by two observers blinded to each group. 1) Hindlimb locomotor function was determined before and after injury and transplantation, us...
- 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
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
Statistical analysis was performed using the software package SPSS 19.0 (IBM Corporation, Armonk, NY, USA).
from paperScoring or quantification
Quantify the primary readouts for this experiment: The anti-inflammatory peptide KAFAKLAARLYRKALARQLGVAA (KAFAK) and brain-derived neurotrophic factor (BDNF)-modified hyaluronan-methylcellulose (HAMC) hydrogel was designed for m...; To explore the profile of KAFAK release by the HAMC hydrogel, we added 25 µL of HAMC-KAFAK/BDNF hydrogels (the concentration of KAFAK: 0.6 µg/mL) to 2 mL sterile PBS....; To explore the profile of BDNF release by HAMC hydrogel, we added 25 µL of HAMC-KAFAK/BDNF hydrogels (the concentration of BDNF: 1 µg/mL) to 2 mL sterile PBS. BDNF rel...; All assessments were performed and analyzed by two observers blinded to each group. 1) Hindlimb locomotor function was determined before and after injury and transplantation, us....
from paperNormalization
Normalize expression or signal values against the stated control or loading reference before comparing groups.
inferred from protocolStatistical comparison
Statistical analysis was performed using the software package SPSS 19.0 (IBM Corporation, Armonk, NY, USA). All data were presented as the mean ± standard error of the mean...; To test the bioactivity of BDNF released from HAMC-KAFAK/BDNF, PC12 cells were cultured with HAMC-KAFAK/BDNF. The cells' proliferation results are shown as a relative rati...; To examine the effect of HAMC-KAFAK/BDNF transplantation on the functional recovery of SCI rats, the BBB rating scale was used to measure locomotor functional recovery during th...; In addition to weekly locomotor evaluation, the inclined plane apparatus was used to assess motor function biweekly ( ). There was no significant difference in the maximal angle...
from paperReporting output
Report representative outputs alongside summary comparisons for The anti-inflammatory peptide KAFAKLAARLYRKALARQLGVAA (KAFAK) and brain-derived neurotrophic factor (BDNF)-modified hyaluronan-methylcellulose (HAMC) hydrogel was designed for m..., To explore the profile of KAFAK release by the HAMC hydrogel, we added 25 µL of HAMC-KAFAK/BDNF hydrogels (the concentration of KAFAK: 0.6 µg/mL) to 2 mL sterile PBS...., To explore the profile of BDNF release by HAMC hydrogel, we added 25 µL of HAMC-KAFAK/BDNF hydrogels (the concentration of BDNF: 1 µg/mL) to 2 mL sterile PBS. BDNF rel..., All assessments were performed and analyzed by two observers blinded to each group. 1) Hindlimb locomotor function was determined before and after injury and transplantation, us....
inferred from protocolStructured statistical methods
Statistical analysis was performed using the software package SPSS 19.0 (IBM Corporation, Armonk, NY, USA). All data were presented as the mean ± standard error of the mean...; To test the bioactivity of BDNF released from HAMC-KAFAK/BDNF, PC12 cells were cultured with HAMC-KAFAK/BDNF. The cells' proliferation results are shown as a relative rati...; To examine the effect of HAMC-KAFAK/BDNF transplantation on the functional recovery of SCI rats, the BBB rating scale was used to measure locomotor functional recovery during th...; In addition to weekly locomotor evaluation, the inclined plane apparatus was used to assess motor function biweekly ( ). There was no significant difference in the maximal angle...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (8)
The anti-inflammatory peptide KAFAKLAARLYRKALARQLGVAA (KAFAK) and brain-derived neurotrophic factor (BDNF)-modified hyaluronan-methylcellulose (HAMC) hydrogel was designed for minimally invasive, localized, and sustained intrathecal protein delivery. The physical and biological characteristics of HAMC-KAFAK/BDNF hydrogel were measured in vitro. SCI model was performed in rats and HAMC-KAFAK/BDNF hydrogel was injected into the injured site of spinal cord. The neuronal regeneration effect was evaluated by inflammatory cytokine levels, behavioral test and histological analysis at 8 weeks post operation.
Maleimide-KAFAK and maleimide-streptavidin were covalently bonded to the MC by thiol-maleimide click chemistry and biotinylated BDNF was conjugated to MC-streptavidin as previously described. Briefly, MC is carboxylated using 1.5 M sodium hydroxide and an overdose of bromoacetic acid. After purification by dialysis, sulfhydryl groups (100 mg of reactive thiols) were integrated into the carboxylated MC polymer (250 mg) main chain upon reaction with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (750 mg) and 3,3′-dithiobis (propionic dihydrazide, 750 mg), followed by reduction of the disulfide bond with dithiothreitol (1.0 g). Maleimide-labeled streptavidin (mol maleimide: mol streptavidin =9:1, 1.1 mg) and maleimide-KAFAK peptide (59 mg) were then added and mixed with previously synthesized sulfhydryl-MC by stirring in 0.1 M PBS (100 mL) overnight at 4°C. N-ethylhydroxy maleimide (13 mg) was then added to quench unreacted free sulfhydryl groups, followed by the removal of unbound maleimide-containing reagent by dialysis. Then MC-KAFAK and MC-streptavidin were sterile-filtered using a 0.22 µm syringe filter and then lyophilized to amorphou...
HAMC-KAFAK/BDNF hydrogels were prepared by physical blending of HA, MC-KAFAK, MC-streptavidin, and biotinylated-BDNF in artificial cerebrospinal fluid (aCSF) with a final composition of 1.5 wt% HA, 2.5 wt% MC-KAFAK, 0.5 wt% MC-streptavidin, and 0.2 wt% biotinylated BDNF. The HAMC hydrogels were prepared with 1.5 wt% HA and 3 wt% MC for comparison. The above ingredients were sterile-filtered before mixing and sequentially added to the aCSF using a high-speed centrifugal mixer (Eppendorf) for 25 seconds at 3,000 rpm/min and dissolved at 4°C for 12 hours. For in vivo studies, the HAMC-KAFAK/BDNF hydrogel compositions were sterilized by filtration and the preparation process was in sterile conditions. The time interval between preparation and use of the final composite hydrogels was less than 15 minutes, during which time the hydrogels were kept at 4°C ( ).
Swelling properties were measured in physiologic conditions. Hydrogels were added into 50 mL centrifuge tubes with PBS at 37°C. At different time points, the hydrogels were taken out, and the weight was measured after removal of excess water on the surface. The swelling ratio (SR) = ( W t - W o )/ W o × 100%. W t is the weight of the swollen hydrogel and W o is the initial weight. The SR was calculated at 6, 12, 18, and 24 hours.
PC12 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM (Gibco ®; Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (Thermo Fisher Scientific), 0.1 mg/mL streptomycin, and 100 U/mL penicillin. Cultures were incubated at 37°C, 5% CO 2, and 95% humidity. The cultivated medium was replaced every 2 days. Cells were passaged when confluence reached 80%-90%. HAMC-KAFAK/BDNF treatment was used to evaluate the effect of released BDNF on PC12 cells proliferation. The PC12 cells were seeded at the density of 1 × 10 4 cells/cm 2 into 96-well plates and 100 µL medium per well. Cells were treated with HAMC-KAFAK/BDNF hydrogels, while the same volume of DMEM was added to the control group. The PC12 cells proliferation was evaluated with a CCK-8 cell proliferation kit (Dojindo Molecular Technologies, Inc., Japan). In brief, at 1, 3, and 7 days, 10 µL CCK-8 solution was applied to each well and incubated for 1 hour, and the absorbance at 450 nm was measured. The experiments were performed in triplicate.
To explore the profile of KAFAK release by the HAMC hydrogel, we added 25 µL of HAMC-KAFAK/BDNF hydrogels (the concentration of KAFAK: 0.6 µg/mL) to 2 mL sterile PBS. KAFAK release into solution was measured using a fluoraldehyde o-phthalaldehyde assay (Thermo Fisher Scientific), by fluorescence analysis. Fluorescence measurements of KAFAK release were taken at 6, 12, 24, 48, 72, and 96 hours. Three replicates were performed.
To explore the profile of BDNF release by HAMC hydrogel, we added 25 µL of HAMC-KAFAK/BDNF hydrogels (the concentration of BDNF: 1 µg/mL) to 2 mL sterile PBS. BDNF release into solution was measured using a rat BDNF ELISA kit (Beijing Solarbio Science & Technology Co., Ltd). Supernatant was collected at 6, 12, 24, 48, 72, and 96 hours. Three replicates were performed. Finally, the accumulated release ratio was calculated.
All procedures were conducted according to protocols approved by Institutional Animal Care and Use Committee of Logistics College of PAP and all experiments were approved by the Institutional Animal Care and Use Committee of Logistics College of PAP. Adult female Sprague Dawley rats (200-230 g) obtained from the Laboratory Animal Center of the Military Medical Science Academy of China were anesthetized with a nitrous oxide/oxygen mixture (70%/30%) containing 1.4% isoflurane delivered by nose cone. All 72 rats were randomized to 4 groups: sham, SCI, HAMC, and HAMC-KAFAK/BDNF (n=18). A laminectomy was performed at the T10 vertebral level to expose the spinal cord and an aneurysm clip with a force of 25 × g was used for 1 minute to develop an SCI model. A HAMC-KAFAK/BDNF or HAMC hydrogel injection was administered after 5 minutes of SCI. A Hamilton syringe was inserted into the center of the traumatic area and 10 µL of the HAMC-KAFAK/BDNF or HAMC hydrogel was injected manually. Following implantation, the muscle and skin of the surgical wound were closed. Animals in the sham group received only the laminectomy without the SCI. The rats were then placed on warming pa...
Machine-readable layer
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"name": "An anti-inflammatory peptide and brain-derived neurotrophic factor-modified hyaluronan-methylcellulose hydrogel promotes nerve regeneration in rats with spinal cord injury methods",
"description": "Evidence-backed execution summary for An anti-inflammatory peptide and brain-derived neurotrophic factor-modified hyaluronan-methylcellulose hydrogel promotes nerve regeneration in rats with spinal cord injury methods from An anti-inflammatory peptide and brain-derived neurotrophic factor-modified hyaluronan-methylcellulose hydrogel promotes nerve regeneration in rats with spinal cord injury.",
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"text": "The anti-inflammatory peptide KAFAKLAARLYRKALARQLGVAA (KAFAK) and brain-derived neurotrophic factor (BDNF)-modified hyaluronan-methylcellulose (HAMC) hydrogel was designed for minimally invasive, localized, and sustained intrathecal protein delivery. The physical and biological characteristics of HAMC-KAFAK/BDNF hydrogel were measured in vitro. SCI model was performed in rats and HAMC-KAFAK/BDNF hydrogel was injected into the injured site of spinal cord. The neuronal regeneration effect was evaluated by inflammatory cytokine levels, behavioral test and histological analysis at 8 weeks post operation."
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"text": "Maleimide-KAFAK and maleimide-streptavidin were covalently bonded to the MC by thiol-maleimide click chemistry and biotinylated BDNF was conjugated to MC-streptavidin as previously described. Briefly, MC is carboxylated using 1.5 M sodium hydroxide and an overdose of bromoacetic acid. After purification by dialysis, sulfhydryl groups (100 mg of reactive thiols) were integrated into the carboxylated MC polymer (250 mg) main chain upon reaction with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (750 mg) and 3,3′-dithiobis (propionic dihydrazide, 750 mg), followed by reduction of the disulfide bond with dithiothreitol (1.0 g). Maleimide-labeled streptavidin (mol maleimide: mol streptavidin =9:1, 1.1 mg) and maleimide-KAFAK peptide (59 mg) were then added and mixed with previously synthesized sulfhydryl-MC by stirring in 0.1 M PBS (100 mL) overnight at 4&#..."
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"@type": "HowToStep",
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"name": "Materials and methods",
"text": "HAMC-KAFAK/BDNF hydrogels were prepared by physical blending of HA, MC-KAFAK, MC-streptavidin, and biotinylated-BDNF in artificial cerebrospinal fluid (aCSF) with a final composition of 1.5 wt% HA, 2.5 wt% MC-KAFAK, 0.5 wt% MC-streptavidin, and 0.2 wt% biotinylated BDNF. The HAMC hydrogels were prepared with 1.5 wt% HA and 3 wt% MC for comparison. The above ingredients were sterile-filtered before mixing and sequentially added to the aCSF using a high-speed centrifugal mixer (Eppendorf) for 25 seconds at 3,000 rpm/min and dissolved at 4°C for 12 hours. For in vivo studies, the HAMC-KAFAK/BDNF hydrogel compositions were sterilized by filtration and the preparation process was in sterile conditions. The time interval between preparation and use of the final composite hydrogels was less than 15 minutes, during which time the hydrogels were kept at 4°C ( )."
},
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"name": "Swelling properties of HAMC-KAFAK/BDNF hydrogels",
"text": "Swelling properties were measured in physiologic conditions. Hydrogels were added into 50 mL centrifuge tubes with PBS at 37°C. At different time points, the hydrogels were taken out, and the weight was measured after removal of excess water on the surface. The swelling ratio (SR) = ( W t - W o )/ W o × 100%. W t is the weight of the swollen hydrogel and W o is the initial weight. The SR was calculated at 6, 12, 18, and 24 hours."
},
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"name": "Cell culture and cell proliferation assay",
"text": "PC12 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM (Gibco ®; Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (Thermo Fisher Scientific), 0.1 mg/mL streptomycin, and 100 U/mL penicillin. Cultures were incubated at 37°C, 5% CO 2, and 95% humidity. The cultivated medium was replaced every 2 days. Cells were passaged when confluence reached 80%-90%. HAMC-KAFAK/BDNF treatment was used to evaluate the effect of released BDNF on PC12 cells proliferation. The PC12 cells were seeded at the density of 1 × 10 4 cells/cm 2 into 96-well plates and 100 µL medium per well. Cells were treated with HAMC-KAFAK/BDNF hydrogels, while the same volume of DMEM was added to the control group. The PC12 cells proliferation was evaluated with a CCK-8 cell proliferation kit (Dojindo Molec..."
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"name": "KAFAK and BDNF release in vitro",
"text": "To explore the profile of KAFAK release by the HAMC hydrogel, we added 25 µL of HAMC-KAFAK/BDNF hydrogels (the concentration of KAFAK: 0.6 µg/mL) to 2 mL sterile PBS. KAFAK release into solution was measured using a fluoraldehyde o-phthalaldehyde assay (Thermo Fisher Scientific), by fluorescence analysis. Fluorescence measurements of KAFAK release were taken at 6, 12, 24, 48, 72, and 96 hours. Three replicates were performed."
},
{
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"position": 7,
"name": "KAFAK and BDNF release in vitro",
"text": "To explore the profile of BDNF release by HAMC hydrogel, we added 25 µL of HAMC-KAFAK/BDNF hydrogels (the concentration of BDNF: 1 µg/mL) to 2 mL sterile PBS. BDNF release into solution was measured using a rat BDNF ELISA kit (Beijing Solarbio Science & Technology Co., Ltd). Supernatant was collected at 6, 12, 24, 48, 72, and 96 hours. Three replicates were performed. Finally, the accumulated release ratio was calculated."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Animals and surgical procedures",
"text": "All procedures were conducted according to protocols approved by Institutional Animal Care and Use Committee of Logistics College of PAP and all experiments were approved by the Institutional Animal Care and Use Committee of Logistics College of PAP. Adult female Sprague Dawley rats (200-230 g) obtained from the Laboratory Animal Center of the Military Medical Science Academy of China were anesthetized with a nitrous oxide/oxygen mixture (70%/30%) containing 1.4% isoflurane delivered by nose cone. All 72 rats were randomized to 4 groups: sham, SCI, HAMC, and HAMC-KAFAK/BDNF (n=18). A laminectomy was performed at the T10 vertebral level to expose the spinal cord and an aneurysm clip with a force of 25 × g was used for 1 minute to develop an SCI model. A HAMC-KAFAK/BDNF or HAMC hydrogel injection was administered after 5 minutes of SCI. A Hamilton syringe was inserted into th..."
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{
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"name": "Swelling properties of HAMC-KAFAK/BDNF hydrogels"
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{
"@type": "HowToTool",
"name": "Animals and surgical procedures"
},
{
"@type": "HowToTool",
"name": "Functional assessments"
},
{
"@type": "HowToTool",
"name": "Quantitative detection of TNF-α, IL-1β, IL-6, and IL-10 by ELISA"
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"name": "Tissue preparation and histochemistry"
},
{
"@type": "HowToTool",
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{
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"name": "HAMC-KAFAK/BDNF promotes behavioral outcomes after SCI"
},
{
"@type": "HowToTool",
"name": "HAMC-KAFAK/BDNF promotes behavioral outcomes after SCI"
}
],
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"name": "Materials and methods"
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{
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"name": "Cell culture and cell proliferation assay"
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{
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"name": "KAFAK and BDNF release in vitro"
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{
"@type": "HowToSupply",
"name": "KAFAK and BDNF release in vitro"
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{
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"name": "Quantitative detection of TNF-α, IL-1β, IL-6, and IL-10 by ELISA"
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{
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"headline": "An anti-inflammatory peptide and brain-derived neurotrophic factor-modified hyaluronan-methylcellulose hydrogel promotes nerve regeneration in rats with spinal cord injury",
"datePublished": "2019",
"author": [
{
"@type": "Person",
"name": "Zhijiang He"
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{
"@type": "Person",
"name": "Hongxin Zang"
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{
"@type": "Person",
"name": "Lei Zhu"
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{
"@type": "Person",
"name": "Kui Huang"
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{
"@type": "Person",
"name": "Tailong Yi"
},
{
"@type": "Person",
"name": "Sai Zhang"
},
{
"@type": "Person",
"name": "Shixiang Cheng"
}
],
"identifier": "10.2147/ijn.s187854"
}
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