During the surgical procedure, there are a few critical steps where the researcher should pay particular attention to in order to obtain optimal outcomes and minimize variability between animals. Care must be taken with the inhaled anesthesia (isoflurane) during surgery as the anesthetic has been shown to have neuroprotective effects with prolonged exposure27. Accordingly, when studying the regenerative capacity of the spinal cord following injury, make an effort to perform the surgery as quickly and efficiently as possible to prevent confounding variables. Maintaining the same isoflurane exposure time per mouse will reduce variability. The breathing rate of the mouse should be monitored throughout the surgery and should not be too slow (less than 1 breath every 2 3 s) or heavily labored (i.e., gasping). The maintenance dose of anesthesia can be lowered from 2 3% to prevent death due to prolonged anesthesia with the note of mice that received the lower dosing.
During the surgery, take extra care when performing muscular incisions on either side of the vertebral column. Ensure that the cuts are deep, and the blade is angled medially so that the edge of the blade rests against the bony vertebrae during muscular incision. If the blade is angled outwards, there is the possibility of excessive bleeding from vascular cuts. The researcher should also pay extra attention when performing the laminectomy to avoid deep angling of the scissors which will damage the spinal cord, causing unwanted tissue damage and functional deficits. The appropriate control for these experiments is a laminectomy only group (no injury), which will enable a comparison of the activation of NSCs attributed to the needle track injury as opposed to that which can result from the laminectomy only. We have shown that laminectomy alone can result in a small, albeit insignificant increase in NSC activation as revealed by an increase in neurosphere numbers from the periventricular region at the level of the lesion21. Laminectomy alone does not cause increases in neurosphere numbers from the periventricular region rostral or caudal to the laminectomy and no neurospheres are found in cultures of white matter isolated at the level of the laminectomy. Additionally, when performing the laminectomy, take care to remove the dorsal lamina in one piece to permit wide exposure of the cord. This will (1) allow sufficient access to perform the minimal needle track injury of the dorsolateral spinal cord, and (2) prevent segments of bone from being left behind and causing secondary damage following closure and post-recovery movement of the mouse. If the entire lamina is not taken as one piece, or segments of sharp bone protruding on either side of the laminectomy are observed, use instruments (such as toothed forceps and curved scissors) to remove these fragments prior to suturing.
The researcher should be extremely careful when applying sutures to the muscular layer during the surgical closure. One suture (double-knotted) should be placed caudal to the level of laminectomy/injury so that the suture lies on top of the intact vertebral bone. This is to prevent any secondary damage that may result from the muscular suture being in contact with the exposed cord when the mouse moves after recovery. Furthermore, the muscular suture caudal to the laminectomy/injury acts as a landmark for where the SCI was performed when isolating the spinal cord for analysis. Care should be taken when dissecting out the injured spinal cord area to avoid compromising the structure of the injured region so that it can remain recognizable during fine dissection under the dissecting microscope. In regard to the neurosphere assay, it is important to perform the mechanical trituration of cell pellets gently to avoid the production of air bubbles that can increase cell death. pNSC-derived neurospheres are even more sensitive to debris heavy, growth conditions excessive trituration and prolonged exposure in enzymatic solutions relative to dNSC cultures. pNSC derived spheres are more compact and smaller than dNSCs. Given the rarity of pNSCs, we recommend isolating at least 240,000 cells per sample.
The minimal SCI model is ideal for studying the cellular events following injury (such as activation of endogenous NSCs) but it does not permit the study of functional impairments. As noted previously, mice that recover from the needle track injury experience no notable behavioral deficits that persist and as such, the mice cannot be evaluated for the effectiveness of therapeutic interventions designed to improve functional outcomes. One important aspect of regenerative medicine is that a treatment should not only promote tissue repair (which can be evaluated using this model, in combination with the neurosphere assay, lineage tracing and immunohistochemistry/immunofluorescence) but should also demonstrate relevant functional improvement using behavioral paradigms where applicable. A number of behavioral tasks used to test functional outcomes in thoracic models of injury such as the foot fault test and the Basso, Beattie, Breshnan (BBB) Open Field Locomotor Scale28, are not sensitive enough to detect measurable deficits in our minimal SCI model. To overcome this shortcoming, one can make use of more sensitive digital scoring systems (e.g., CatWalk) which measures multiple parameters of gross and fine hind-limb locomotion parameters including gait analyses, which may detect the minimal deficits resulting from the minimal injury model29.
This method could also be adapted to study endogenous NSC activation as a potential therapy in models of cervical spinal cord injury. Cervical SCI is the most clinically relevant model and we propose that adapting this injury model to higher vertebral segments would provide insight into whether there are regional variations in the response of NSCs and their progeny (kinetics, migration, differentiation) and whether the lesion would result in more profound and measurable functional deficits. The currently used murine cervical injury models (such as transection, clip compression and/or contusion) require intensive post-operative care including the need to manually express bladders and constantly monitor the breathing of the mouse. Adapting the minimal injury model to the cervical spinal cord may reduce the mortality, morbidity and post-operative care associated with other cervical SCI models as well as permit one to examine the effectiveness of drugs/small molecules and/or rehabilitation outcomes on neural recovery. Our injury model can also be adapted to create a minimal injury in different parts of the cord (i.e., dorsal or lateral columns) and/or larger injuries with deeper penetration and/or the use of larger sized needles (smaller gauge). This allows the researcher to control/manipulate the type and size of the lesion and thus evaluate cellular and functional/behavioral recovery accordingly.
The minimal injury model in mice permits the use of transgenic animal models. The mouse models enable labeling of endogenous stem cells and/or progenitors prior to the injury. This can allow the researcher to track the fate of these pre-labeled cells following injury and evaluate neural precursor cell proliferation, migration, and differentiation following injury potentially contributing to neural repair.
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