Neurosphere culture derived from aged hippocampal dentate gyrus.

The neurosphere assay is the gold standard for determining proliferative and differentiation potential of neural progenitor cells (NPCs) in neurogenesis studies 1-3 . While several in vitro assays have been developed to model the process of neurogenesis, they have predominantly used embryonic and early postnatal NPCs derived from the dentate gyrus (DG). A limitation of these approaches is that they do not provide insight into adult-born NPCs, which are modeled to affect hippocampal function and diseases later in life. Here, we show a novel free-floating neurosphere culture system using NPCs isolated from the DG of mature adult and aged mice. The protocol outlines detailed steps on the isolation, propagation, and maintenance of neurospheres from adult and aged (>12 months old) mouse brain and how to differentiate cultured neurospheres into neurons and astrocytes. Culturing adult and aged NPCs provides an important in vitro model to (1) investigate cellular and molecular properties of this unique cell population and (2) expand the understanding of plasticity in the adult and aging brain. This protocol requires ∼2 hours to complete dissection, dissociation and culture plating, while differentiation to neuronal and astrocytic lineages takes 9 days. By focusing on neurospheres obtained from animals at later ages this model facilitates investigation of important biological questions related to development and differentiation of hippocampal neurons generated throughout adult life.

Evaluating the performance of OCT in assessing static and potential dynamic properties of the retinal ganglion cells and nerve fiber bundles in the living mouse eye.

Glaucoma is a group of eye diseases characterized by the thinning of the retinal nerve fiber layer (RNFL), which is primarily caused by the progressive death of retinal ganglion cells (RGCs). Precise monitoring of these changes at a cellular resolution in living eyes is significant for glaucoma research. In this study, we aimed to assess the effectiveness of temporal speckle averaging optical coherence tomography (TSA-OCT) and dynamic OCT (dOCT) in examining the static and potential dynamic properties of RGCs and RNFL in living mouse eyes. We evaluated parameters such as RNFL thickness and possible dynamics, as well as compared the ganglion cell layer (GCL) soma density obtained from in vivo OCT, fluorescence scanning laser ophthalmoscopy (SLO), and ex vivo histology.

Fidgetin-like 2 negatively regulates axonal growth and can be targeted to promote functional nerve regeneration.

The microtubule (MT) cytoskeleton plays a critical role in axon growth and guidance. Here, we identify the MT-severing enzyme fidgetin-like 2 (FL2) as a negative regulator of axon regeneration and a therapeutic target for promoting nerve regeneration after injury. Genetic knockout of FL2 in cultured adult dorsal root ganglion neurons resulted in longer axons and attenuated growth cone retraction in response to inhibitory molecules. Given the axonal growth-promoting effects of FL2 depletion in vitro, we tested whether FL2 could be targeted to promote regeneration in a rodent model of cavernous nerve (CN) injury. The CNs are parasympathetic nerves that regulate blood flow to the penis, which are commonly damaged during radical prostatectomy (RP), resulting in erectile dysfunction (ED). Application of FL2-siRNA after CN injury significantly enhanced functional nerve recovery. Remarkably, following bilateral nerve transection, visible and functional nerve regeneration was observed in 7 out of 8 animals treated with FL2-siRNA, while no control-treated animals exhibited regeneration. These studies identify FL2 as a promising therapeutic target for enhancing regeneration after peripheral nerve injury and for mitigating neurogenic ED after RP - a condition for which, at present, only poor treatment options exist.