GSE109902
Transcriptional screen in the target region of sprouting hindlimb corticospinal fibers after thoracic spinal cord injury in rats
Depending on the severity of a CNS injury, in humans as well as in experimental animal models certain degrees of functional recovery are observed. Rodent studies have provided much evidence for underlying anatomical changes involving neurite outgrowth of both spared and axotomized fiber tracts, also referred to as sprouting. Following a thoracic spinal cord injury (SCI) in adult rats, axotomized fibers of the hindlimb corticospinal tract (CST) were found to sprout into the cervical spinal cord at different rostrocaudal levels. Anterograde tracings showed that these new collaterals of motor cortical hindlimb fibers project into the intermediate laminae of the cervical grey matter, particularly pronounced at the level of cervical segment three (C3). The molecular cues which induce the growth and guide these sprouts of hindlimb CST fibers are unknown. We hypothesize that sectreted factors and guidance and adhesion molecules are specifically expressed in the intermediate laminae of the cervical spinal cord to direct and target the ingrowth of the sprouting hindlimb CST fibers. To test our hypothesis the transcription profile of the target region of sprouts was investigated with RNA sequencing of total RNA extracted from the intermediate laminae (IV-VII) of the spinal gray matter at cervical level C3. Four major comparisons were performed, which were then again compared to each other. The first comparison was between samples of animals with an SCI sacrificed at four different time points after injury (1, 3, 6, 12 weeks after injury). The second and third comparison was between samples of rats with a SCI compared to their sham controls sacrificed at the same time point after surgery (1 week or 12 weeks respectively). The fourth comparison was between samples from animals of the two control groups, rats with a sham surgery which were either sacrificed at one or at twelve weeks after injury. We found that most changes in gene expression ocurred at one week after SCI and least changes occured at twelve weeks after SCI, suggesting that the largest effects on a global level of gene expression occur during early time points after injury. Interestingly, also we also found changes in gene expression when comparing the samples from the two control groups, which suggests that sham surgery consisting of a laminectomy is sufficient to induce transcriptional changes in the spinal cord. This is the first study to provide insight into transcriptional changes in the target region of sprouts investigating the role of the cervical spinal cord (target region) for sprouting of hindlimb CST fibers. Overall design: Experiments were started with a total of 67 adult female Long Evans rats (250-300 g, obtained from Charles River Germany), which were housed in groups of four in a 12:12 hours light/dark cycle. Food and water were available ad libitum. Animals were given at least one week to acclimatize before the experimentation began. All experiments were performed during the light cycle. All animal procedures were conducted in accordance with the Veterinary Office of Canton Zurich, Zurich, Switzerland. Animals were stratified based on their baseline performance on the Montoya staircase test and then randomly assigned to 6 groups: n=10 in the control group euthanized at 1 week after sham surgery, n=10 in the control group euthanized at 12 weeks after sham surgery, n=10 in the SCI group euthanized at 1 week after injury (1 week group), n=10 in the SCI group euthanized at 3 weeks after injury (3 weeks group). n=11 in the SCI group euthanized at 6 weeks after injury (6 weeks group), and 6) n=16 in the SCI group euthanized at 12 weeks after injury (12 weeks group). Due to inadequate lesion size five animals were excluded. Based on RNA quality and quantity five samples per group were chosen for RNA sequencing (total  sample size= 30, 5 biological replicates/condition).