Injured retinal ganglion cell (RGCs) axons do not regenerate spontaneously, causing loss of vision in glaucoma and after trauma. Recent studies have identified several strategies that induce long distance regeneration in the optic nerve. Thus, a pressing question now is whether regenerating RGC axons can find their appropriate targets. Traditional methods of assessing RGC axon regeneration use histological sectioning. However, tissue sections provide fragmentary information about axonal trajectory and termination. To unequivocally evaluate regenerating RGC axons, here we apply tissue clearance and light sheet fluorescence microscopy (LSFM) to image whole optic nerve and brain without physical sectioning. In mice with PTEN/SOCS3 deletion, a condition known to promote robust regeneration, axon growth followed tortuous paths through the optic nerve, with many axons reversing course and extending toward the eye. Such aberrant growth was prevalent in the proximal region of the optic nerve where strong astroglial activation is present. In the optic chiasms of PTEN/SOCS3 deletion mice and PTEN deletion/Zymosan/cAMP mice, many axons project to the opposite optic nerve or to the ipsilateral optic tract. Following bilateral optic nerve crush, similar divergent trajectory is seen at the optic chiasm compared to unilateral crush. Centrally, axonal projection is limited predominantly to the hypothalamus. Together, we demonstrate the applicability of LSFM for comprehensive assessment of optic nerve regeneration, providing in-depth analysis of the axonal trajectory and pathfinding. Our study indicates significant axon misguidance in the optic nerve and brain, and underscores the need for investigation of axon guidance mechanisms during optic nerve regeneration in adults.

A variety of in vitro models has been developed to understand the mechanisms underlying the regenerative failure of central nervous system (CNS) axons, and to guide pre-clinical development of regeneration-promoting therapeutics. These range from single-cell based assays that typically focus on molecular mechanisms to organotypic assays aimed at recapitulating in vivo behavior. By utilizing a combination of models, researchers can balance the speed, convenience, and mechanistic resolution of simpler models with the biological relevance of more complex models. This review will discuss a number of the models that have been used to build our understanding of molecular mechanisms of CNS axon regeneration.

Astrocyte phenotypes change in a process called reactive gliosis after traumatic central nervous system (CNS) injury. Astrogliosis is characterized by expansion of the glial fibrillary acidic protein (GFAP) cytoskeleton, adoption of stellate morphologies, and differential expression of some extracellular matrix molecules. The astrocytic response immediately after injury is beneficial, but in the chronic injury phase, reactive astrocytes produce inhibitory factors ( i.e., chondroitin sulfate proteoglycans [CSPGs]) that limit the regrowth of injured axons. There are no drugs that promote axon regeneration or functional recovery after CNS trauma in humans. To develop novel therapeutics for the injured CNS, we screened various libraries in a phenotypic assay to identify compounds that promote neurite outgrowth. However, the effects these compounds have on astrocytes are unknown. Specifically, we were interested in whether compounds could alter astrocytes in a manner that mimics the glial reaction to injury. To test this hypothesis, we developed cell-based phenotypic bioassays to measure changes in (1) GFAP morphology/ localization and (2) CSPG expression/immunoreactivity from primary astrocyte cultures. These assays were optimized for sixpoint dose-response experiments in 96-well plates. The GFAP morphology assay is suitable for counter-screening with a Zfactor of 0.44 -0.03 (mean -standard error of the mean; N = 3 biological replicates). The CSPG assay is reproducible and informative, but does not satisfy common metrics for a ''screenable'' assay. As proof of principle, we tested a small set of hit compounds from our neurite outgrowth bioassay and identified one that can enhance axon growth without exacerbating the deleterious characteristics of reactive gliosis.

Object. Simultaneous traumatic brain injury (TBI) and aortic injury has been considered unsurvivable for many years because treatments such as sedation and blood pressure goals conflict for these 2 conditions. Additionally, surgical interventions for aortic injury often require full anticoagulation, which is contraindicated in patients with TBI. For these reasons, and due to the relative rarity of aortic injury/TBI, little data are available to guide treating physicians.Methods. A retrospective review was performed on all simultaneous TBI and aortic injury cases from 2000 to 2012 at a university-affiliated, Level I trauma center. Patient demographics, imaging studies, interventions, and outcomes were analyzed. Traumatic brain injury/aortic injury cases treated with endovascular stenting were specifically studied to determine trends in procedure timing, use of anticoagulation, and neurological outcome.Results. Thirty-three patients with concurrent TBI and aortic injury were identified over a 12-year period. The median patient age was 44 years (range 16-86 years) and the overall mortality rate after imaging diagnosis was 46%. All surviving patients were awake and neurologically functional at discharge, and 83% were discharged home or to rehabilitation facilities. Patients who died had a higher Injury Severity Scale score (p = 0.006). Severe TBI (p = 0.045) or hemodynamic instability (p = 0.015) upon arrival to the hospital was also correlated with increased mortality rates. Thirty-three percent of aortic injury/TBI patients (n = 11) underwent endovascular stenting, and 7 of these patients received intravenous anticoagulation therapy at the time of surgery. Six of these 7 anticoagulation-treated patients experienced no significant progression on postoperative brain CT, whereas 1 patient died of hemodynamic instability prior to undergoing further imaging.Conclusions. Simultaneous TBI and aortic injury is a rare condition with a historically poor prognosis. However, these results suggest that many patients can survive with a good quality of life. Technological advances such as endovascular aortic stenting may improve patient outcome, and anticoagulation is not absolutely contraindicated after TBI. Key woRDS • aortic injury • traumatic brain injury • aortic stentAbbreviations used in this paper: GCS = Glasgow Coma Scale; ISS = Injury Severity Scale; SAH = subarachnoid hemorrhage; SDH = subdural hematoma; TBI = traumatic brain injury.