Mice
Young adult male and female mice were used between 2 and 4 months of age at the time of experimental procedures. C57BL/6 J mice (JAX: 000664) were used for experiments requiring a wild-type background. For RNA-seq of astrocyte-specific ribosome-associated mRNA mice expressing RiboTag43 (JAX: 029977) were crossed to the well-characterized, astrocyte-specific Cre-driver line, mGfap-cre 73.1237 to generate mGfap-cre-RiboTag mice. mGfap-cre-RiboTag mice were crossed to Stat3-loxP mice10 to generate mGfap-cre-Ribotag-Stat3-loxP mice (Stat3-cKO). Astrocyte-conditional Ccn1-knockout mice were obtained by crossing the well-characterized, astrocyte-specific Cre-driver line Aldh1l1-CreERT2 (ref. 31) JAX: 031008 to the Ccn1-LoxP line32 (a gift from K. Lyons) to generate Ccn1-cKO mice. Aldh1l1-CreERT2 mice were crossed to the floxed-STOP-tdT (Ai9) reporter line to generate Aldh1l1-CreERT2::floxed-STOP-tdT mice. Cre recombinase expression was activated in young adult mice (6–8 weeks old) by administering tamoxifen (Sigma, T5648-1G, 20 mg ml−1 in corn oil) by subcutaneous injection (100 mg kg−1, once a day) for 5 days followed by clearance for 3 weeks so that no residual tamoxifen remained at the time of experiment initiation. All mice were housed in a facility with a 12 h:12 h light:dark cycle and controlled temperature and humidity, and were allowed free access to food and water. Experiments were conducted according to protocols approved by the Institutional Animal Care and Use Committee at Cedars-Sinai medical centre.
Surgical procedures
All surgeries were performed on male and female young adult mice (8–12 weeks old) under general anaesthesia with isoflurane in oxygen-enriched air using an operating microscope (Zeiss), and rodent stereotaxic apparatus (David Kopf).
Spinal cord injury
Laminectomy of a single vertebra was performed at spinal cord level T12. Incomplete iSCI by unilateral T12 hemisection was performed on the left side of the spinal cord using a microknife (Fine Science Tools). To be included in the study, mice exhibited complete unilateral hindlimb paralysis for the first three days following surgery. A T12 crush SCI was made using no. 5 Dumont forceps (Fine Science Tools) with a 0.4 mm spacer and with a tip width of 0.5 mm. T12 crush mice exhibited paralysis in both hind limbs. In each case, mice received the opiate analgesic buprenorphine subcutaneously before surgery and every 12 h for 48 h after injury. Mice were evaluated thereafter blind to genotype and experimental condition. Daily bladder expression was performed for the duration of the study or until voluntary voiding returned.
Injections of lysolecithin or myelin into the spinal cord
Five-hundred nanolitres of 1% lysolecithin or 1 mg ml−1 CFSE-myelin in PBS was delivered by intervertebral microinjection to the lateral spinal cord white matter at spinal cord level T12 (coordinates: 200 μm medial–lateral, 300 μm dorsal–ventral). Injections were carried out at 150 nl min−1 using finely bevelled glass micropipettes connected via high-pressure tubing (Kopf) to 10 μl gastight syringes under the control of microinfusion pumps (Harvard Apparatus). Needles were left in place for 6 min prior to being slowly retracted. An equal volume of PBS was injected into the contralateral white matter as vehicle control. Mice were euthanized at 3 days post myelin injection and at 3, 10 and 25 days post-lysolethicin.
Sciatic nerve injury
A small incision was made on the left hindlimb and the two heads of the bicep femoris muscle were gently separated to reveal the sciatic nerve. The sciatic nerve was released from the muscle and elevated using forceps. The isolated nerve was then clamped with haemostats for 10 s and then replaced under the muscle. Mice were euthanized seven days following sciatic nerve crush.
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