Axonal degeneration is a key component of neurodegenerative diseases such as Huntington’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis (ALS). (NAm), an NAD+ precursor, has long since been implicated in axonal protection and reduction of degeneration. On the other hand, hydrogen peroxide (H2O2) has been implicated in oxidative stress and axonal degeneration. The effects of laser-induced axonal damage in wild-type (WT) and Huntington’s disease(HD) mouse dorsal root ganglion neurons (DRGs) treated with NAm or H2O2 were investigated and the cell body width, axon width, axonal strength, and axon shrinkage post laser-induced injury were measured. We found that HD mouse DRGs have increased strength against laser damage compared to wild-type DRGs. We additionally found that treatment with NAm reduces the neuronal strength against laser damage in both WT and HD DRGs. Interestingly, when comparing HD DRGs treated with H2O2 and WT DRGs treated with H2O2, we found that treatment with H2O2 reduced the time required for the RoboLase laser system to cut through HD DRGs. We additionally found that both NAm and H2O2 treatments resulted in morphological changes in both WT and HD DRG cell bodies, respectively. We did not find any difference in shrinkage across the models. Ultimately, our results suggest that H2O2 at the same concentration may have less damaging effects on WT neurons than previously expected. Our results additionally indicate that higher concentrations of NAm, previously deemed to be safe, may have a neurotoxic effect rather than an axonal protective effect on HD and WT DRGs.
Neuronal responses to injury are of interest to the development of methods to mitigate damage and stimulate repair. We utilized a single pulse from a 1030nm laser to create a laser induced shockwave (LIS) to subject neuronal cells to injury and compare the effects of injury on neurons from an Alzheimer’s Disease (AD) mouse model and wild type (WT) mice. We found differences in the calcium response to LIS in AD versus WT neurons. Additionally, we found that LIS induced cell death led to a calcium elevation which differed from that in cells that stayed alive. Therefore, the calcium response can be utilized to separate dead cells from live cells.
Laser induced shockwave (LIS) can be utilized to subject neuronal cells to conditions similar to those occurring during a blast induced traumatic brain injury. We utilized a 532nm Coherent Flare laser to induce a shockwave near cells which had been transfected with a FRET calcium biosensor (D3CPV) so that we could monitor the immediate cellular responses. Our shockwave system was characterized with a high-speed camera to monitor cavitation bubble dynamics and calculate the shear forces cells were subjected to. We found that we could induce forces which have been previously shown to induce injury. Using both phase and fluorescence microscopy we monitored the effects of shear on our cells. We found that at distances up to 120 microns from the laser focal point cells experienced shears greater than 10kPa. At those distances cell fragmentation was observed. Cells that survived and expressed the FRET biosensor demonstrated an immediate calcium elevation irrespective of extracellular or cytoplasmic calcium concentration. Cells recovered to pre-shockwave calcium levels within ~30s. In conclusion, LIS can be utilized to simultaneously monitor the neuronal response to shear stress and nearby cell death or injury.
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