All animal care and surgical interventions were undertaken in strict accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals, Guide for the Care and Use of Laboratory Animals, and with the approval of Animal Welfare Committee at the University of Texas Health Science Center at Houston.
A total of 24 female C57BI/6J mice (10-16 weeks of age; 20-25g; The Jackson Laboratory, Bar Harbor, ME) were used with 3 mice pooled together in each biological replicate: sham control (n=2), 2 and 7 days after SCI (n=3). The surgical procedure for SCI were described previously [28 (link)]. Briefly, after anesthetization with a mixed solution of ketamine (80 mg/kg ip) and xylazine (10 mg/kg ip), mice received a dorsal laminectomy at the 9th thoracic vertebral (T9) level to expose the spinal cord and then a moderate T9 contusive injury using an Infinite Horizons impactor (Precision Systems and Instrumentation) at 60 kdyn with the spine stabilized using steel stabilizers inserted under the transverse processes one vertebra above and below the injury [29 (link)]. The sham control mice received only a dorsal laminectomy without contusive injury. Afterwards, the wound was sutured in layers, bacitracin ointment (Qualitest Pharmaceuticals, Huntsville, AL) was applied to the wound area, 0.1mL of a 20 mg/ml stock of gentamicin (Butler Schein, Dublin, OH) was injected subcutaneously, and the animals recovered on a water-circulating heating pad. Then mice received analgesic agent, buprenorphine(0.05 mg/kg, SQ; Reckitt Benckise, Hull, England) twice a day for two days. Bladders were manually expressed until automatic voiding returned spontaneously, which generally was within 7 days. The mice locomotion tests, Basso Mouse Score, were performed at 2 and 7 days post-injury before collecting the injured spinal cord tissues to confirm the injury severity in each mouse is consistent with moderate contusion SCI. At 2 or 7 days after SCI, the mice were anesthetized again with ketamine and xylazine and perfused briefly with normal physical saline. The injured spinal cords were then dissected. 0.5 mm pieces of spinal cord were cut in the injured epicenter and frozen in liquid nitrogen and processed for RNA isolation. Histological staining was done by the iron-eriochrome cyanine R (EC) staining. The spinal cords from the epicenter, 6mm and 12mm to the epicenter caudally and rostrally were used for staining.
A total of 24 female C57BI/6J mice (10-16 weeks of age; 20-25g; The Jackson Laboratory, Bar Harbor, ME) were used with 3 mice pooled together in each biological replicate: sham control (n=2), 2 and 7 days after SCI (n=3). The surgical procedure for SCI were described previously [28 (link)]. Briefly, after anesthetization with a mixed solution of ketamine (80 mg/kg ip) and xylazine (10 mg/kg ip), mice received a dorsal laminectomy at the 9th thoracic vertebral (T9) level to expose the spinal cord and then a moderate T9 contusive injury using an Infinite Horizons impactor (Precision Systems and Instrumentation) at 60 kdyn with the spine stabilized using steel stabilizers inserted under the transverse processes one vertebra above and below the injury [29 (link)]. The sham control mice received only a dorsal laminectomy without contusive injury. Afterwards, the wound was sutured in layers, bacitracin ointment (Qualitest Pharmaceuticals, Huntsville, AL) was applied to the wound area, 0.1mL of a 20 mg/ml stock of gentamicin (Butler Schein, Dublin, OH) was injected subcutaneously, and the animals recovered on a water-circulating heating pad. Then mice received analgesic agent, buprenorphine(0.05 mg/kg, SQ; Reckitt Benckise, Hull, England) twice a day for two days. Bladders were manually expressed until automatic voiding returned spontaneously, which generally was within 7 days. The mice locomotion tests, Basso Mouse Score, were performed at 2 and 7 days post-injury before collecting the injured spinal cord tissues to confirm the injury severity in each mouse is consistent with moderate contusion SCI. At 2 or 7 days after SCI, the mice were anesthetized again with ketamine and xylazine and perfused briefly with normal physical saline. The injured spinal cords were then dissected. 0.5 mm pieces of spinal cord were cut in the injured epicenter and frozen in liquid nitrogen and processed for RNA isolation. Histological staining was done by the iron-eriochrome cyanine R (EC) staining. The spinal cords from the epicenter, 6mm and 12mm to the epicenter caudally and rostrally were used for staining.
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