INJS6
Sus scrofa
Protocols: Prior to euthanasia, anesthesia was induced using Telazol (4 mg/Kg) and Xylazine (2 mg/kg) followed by Propofol (3-7 mg/kg) and then maintained with Isoflurane (1-3 MAC) following intubation. Eyes were protected using an eye lubricant. Both limbs were shaved and scrubbed with Chlorhexidine and 70% alcohol until visibly clean, followed by a ten minute evaporation period. Hoofs were covered with unsterile gloves. Animals were then transferred into the adjacent operating room, placed supine on a heating mat, and secured on the operation table. Animal health and anesthesia depth were maintained by monitoring respiratory rate, oxygen saturation, electrocardiogram, blood pressure, and body temperature. The surgical limb and ipsilateral lower body were then scrubbed three times using Betadine. Hoofs were covered with a sterile glove and secured using a sterile elastic wrap. One layer of sterile towels was placed around the surgical area, followed by a layer of sterile drapes, leaving only the surgical limb exposed during the procedure. Animals were euthanized during deep anesthesia using an intravenous injection of a solution containing pentobarbital sodium and phenytoin sodium (Beuthanasia-D, 0.1ml/kg). Death was confirmed by the absence of blood pressure and heart sounds prior to obtaining the tissue samples. Cartilage samples were harvested from the medial femoral condyle for RNA isolation and histopathological analysis. The cartilage posterior to the frontal plane in the center of the medial femoral condyle was allocated to RNA isolation, while the cartilage immediately anterior of the central plane was allocated to histology. Biopsy punches of 5 mm diameter (Miltex, York, PA) were used to obtain 4-8 osteochondral biopsies from each animal. Osteochondral samples were rinsed in water and the cartilage was separated from the subchondral bone. Cartilage samples were immediately frozen in liquid nitrogen and stored at -80°C until RNA isolation. Forty-two adolescent Yucatan minipigs (Sinclair BioResources, Columbia MO) were allocated to unilateral ACL transection surgery (n=36) or no surgery (INTACT, n=6). Animals receiving ACL transection surgery were allocated to euthanasia and outcome assessment at 1 week (1W, n=18) or 4 weeks (4W, n=18) after surgery. Within each time-point 6 of 18 animals were allocated to no treatment following transection, 6 of 18 to immediate ligament reconstruction surgery, and 6 of 18 to immediate ligament repair surgery. A computer based random permutation stratified for sex determined each animal's group allocation and side of unilateral surgery with an equal number of males and females in each group. While the animals were under anesthesia, a medial arthrotomy was performed and the fat pad partially resected to expose the ACL. The ACL was transected between the proximal and middle thirds of the ligament. A clinical exam was performed to verify complete ACL transection. In the animals assigned to receive ACL reconstruction surgery, a fresh-frozen bone-patellar-tendon-bone allograft, which was harvested from an age-, weight-, and sex-matched donor, was implanted as previously described (Murray and Fleming. AJSM 2013). In the animals assigned to ACL repair surgery, an extracellular matrix scaffold in combination with autologous blood was implanted as previously described (Murray and Fleming. AJSM 2013). Knees were thoroughly irrigated with 500 mL of normal saline following ACL transection in the group that did not receive further surgical treatment or following bone tunnel placement in those that received ACL reconstruction or repair. All incisions were closed in layers. No postoperative immobilization was used. Total RNA was extracted from the synovial membranes through homogenization, phenol-chloroform separation and on-column purification using the PureLink™ RNA Mini Kit (Life technologies, Carlsbad, CA). RNA samples were treated with DNAse I (PureLink™ DNase Set, Life technologies, Carlsbad, CA) and assessed with the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). The samples were then enriched for polyA+ messenger RNA, reverse transcribed with random hexamers, ligated with indexed adapters and amplified with 17 cycles of PCR using the TruSeq RNA Sample Preparation Kit v2 (Illumina, San Diego, CA).
ENA first public
2017-10-22
ENA last update
2017-07-31
External Id
SAMEA104188425
INSDC center alias
'Boston Children''s Hospital, Harvard Medical School'
INSDC center name
'Boston Children''s Hospital, Harvard Medical School'
INSDC first public
2017-10-22T17:02:54Z
INSDC last update
2017-07-31T15:29:38Z
INSDC status
public
Submitter Id
E-MTAB-5879:INJS6
age
18.7
breed
Yucatan
broker name
ArrayExpress
common name
pig
organism part
cartilage
sample name
E-MTAB-5879:INJS6
sampling site
medial condyle of femur
sex
female