Spared nerve injury surgery was performed as described (Decosterd and Woolf, 2000 (link)). Briefly, mice were anesthetized with a mix of ketamine/xylazine while an incision was made through the skin and thigh muscle at the level of the trifurcation of the sciatic nerve. The common tibial and peroneal nerve were ligated with 6.0 silk (Ethicon) and transected, leaving the sural nerve intact. Mechanical thresholds were determined 7 days after the surgery, before and after drug administration, and 30 days after the surgery. Mechanical threshold was determined using the up-down method.
6 0 silk
Manufactured by Johnson & Johnson
6-0 silk is a medical-grade surgical suture material made of natural silk fibers. It is a thin, monofilament suture used in delicate surgical procedures. The suture is designed to provide strength and durability while maintaining minimal tissue reaction.
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9 protocols using 6 0 silk
1
Spared Nerve Injury Surgery Protocol
2
Neuropathic Pain Modeling via Spared Nerve Injury
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Fourteen days after intrathecal NPY-saporin or blank-saporin injection, animals underwent SNI surgery. SNI was performed as described previously9 (link),26 (link). Anesthesia was induced and maintained with 5% and 2–3% isoflurane, respectively. After shaving and Betadine wipe of the left hind limb, an incision was made in the skin at the level of the trifurcation of the left sciatic nerve. The overlying biceps femoris muscles were retracted, exposing the tibial, common peroneal, and sural nerve branches. The common peroneal and tibial nerves were ligated with 6-0 silk (Ethicon, Somerville, NJ), and then the knot and adjacent nerve (2 mm) were transected, leaving the sural branch intact. The muscle was sutured with 4-0 silk sutures and the wound was closed with 9-mm metal clips, followed by Neosporin®.
3
Gelatin Hydrogel-Based Vascular Bed
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The vascular bed was created using Male Jcl:SD rats weighing 270–470 g (Clea Japan). A bioabsorbable Gelatin hydrogel (Nitta Gelatin) was cut into 12 mm × 12 mm pieces and incubated in 200 mL PBS containing/not containing cytokines (1.0 μg bFGF and 0.01 μg VEGF; Nacalai Tesque) at 4 °C for 24 h. The Jcl:SD rat was anesthetized by inhalation of 2% isoflurane, and the SFA and SFV were partially isolated from the surrounding tissue. A porous EVOH membrane (supplied by Kuraray) was placed under the SFA and SFV, and the Gelatin gel (prepared the day before) was transplanted onto the SFA and SFV. The EVOH membrane was folded over the top of the Gelatin sheet, and the edges of the EVOH membrane were sealed with heat to encapsulate the Gelatin sheet and underlying blood vessels. Two weeks later, the SFA was ligated distal to the Gelatin sheet using 6-0 silk (Ethicon). The vascular bed created in the gel was evaluated after an additional week of incubation, and the rat was then euthanized with an overdose of pentobarbital.
4
Rodent Sciatic Nerve Injury Model
5
Xenografting Ovarian Tissue Cryopreservation
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Xenografting of fresh and cryopreserved tissue was performed at the Cornell University laboratory as described previously [17 (link)]. All tissue fragments from different conditions, that is, fresh (18 mice, 214 grafts), uncontrolled slow (15 mice, 210 grafts) or vitrification (16 mice, 222 grafts) were xenografted to different recipient mice. Briefly, anesthesia was induced and then maintained in the male SCID mice using 1.5 to 3.5% isoflurane gas. Castration was performed via a midline abdominal approach to eliminate endogenous gonadal hormone influence. Then, three to six, 1 cm long incisions were made in the skin over the back to insert 11 to 14 tissue pieces (xenografts) bilaterally (5–7 pieces per side). A 6–0 silk (Ethicon, Somerville, NY) suture was placed to mark the site of each graft to facilitate subsequent piece recovery. The incisions were closed with skin staples (Braintree Scientific, Braintree, MA) and an i.m. injection of buprenorphine (Bedford Laboratories, Bedford, OH; 1 mg kg-1) provided for analgesia. For graft recovery, each mouse was euthanized by cervical dislocation 17 wk after graft placement. A dorsal skin incision was made to expose the grafts that were excised and immediately immersed in Bouin’s fixative solution.
6
Mouse Hindlimb Ischemia Model and Stem Cell Transplantation
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The 6-week-old female nude mice (body weight 25-30 g) (Orient bio) were anesthetized with rompun (20 mg/kg) and ketamine (100 mg/kg). The femoral artery and its branches were ligated through a skin incision with a 6-0 silk (Ethicon). The external iliac artery and all of the above arteries were then ligated. The femoral artery was excised from its proximal origin as a branch of the external iliac artery to the distal point where it bifurcates into the saphenous and popliteal arteries. Prior to transplantation, cells were labeled with CM-DiI (DiI, Molecular Probes) to detect survival and engraftment in vivo. The number of mES-ECs (1 × 105 cells/mouse) were suspended in 200 μl of PBS then injected intramuscularly into four sites of the gracilis muscle in the medical thigh with a 29-gauge insulin syringe.
7
Spared Nerve Injury Mouse Model
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Nerve injury was induced using the SNI model of neuropathic pain, as adapted for mice14 (link),60 (link) at 10 to 12 weeks of age. Under deep isoflurane anesthesia, an incision was made on the medial surface of the thigh, exposing the 3 branches of the sciatic nerve. Spared nerve injury surgery consisted of ligation and transection of the left tibial and common peroneal branches of the sciatic nerve, while sparing the sural nerve. The tibial and common peroneal branches were tightly ligated with 6:0 silk (Ethicon, Cincinnati, OH) and sectioned distal to the ligation. Sham surgery consisted of exposing the nerve without damaging it.
8
Spared Nerve Injury Mouse Model
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Animals were randomly assigned to receive either spared nerve injury (SNI) or sham surgery. Nerve injury was induced using the SNI model of neuropathic pain, as adapted for mice18 (link),59 (link) at 10 to 12 weeks of age. Under deep isoflurane anesthesia, an incision was made on the medial surface of the thigh, exposing the 3 branches of the sciatic nerve. The tibial and common peroneal branches were tightly ligated with 6-0 silk (Ethicon) and sectioned distal to the ligation. Sham surgery consisted of exposing the nerve without damaging it.
9
Implantation of Bioengineered Muscle Constructs
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In vitro regenerated muscle constructs (n=6) were transplanted into 6-weeks old, female Nude mice. Under general anesthesia, a 1.5 cm skin incision was performed on the anterolateral side of the lower hind limb and the fascia of the tibialis anterior muscle was carefully lifted from the underlying muscle. The regenerated muscle construct was then placed between fascia and muscle, thereby matching the fiber direction of the native muscle. (12) The skin incision was closed with 6-0 silk (Ethicon), the animals were weaned from anesthesia and single housed. Animals receiving regenerated muscle implants showed no signs of limping or reduced physical activity on day 1 after implantation.
Bioengineered muscle constructs were explanted from sacrificed animals on day 14 after implantation, fixed with PFA and embedded in paraffin for histology.
Bioengineered muscle constructs were explanted from sacrificed animals on day 14 after implantation, fixed with PFA and embedded in paraffin for histology.
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