Introduction

Neurodegenerative diseases, such as amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease, share morphological features of axonal degradation that precede the death of the cell body as an early event in disease (1,2,3). Presently, there are limited methods to quantify protection afforded by candidate molecules that may prevent, delay, or slow the degradation process. Current methods for quantifying neurodegeneration—such as measurement of longest remaining neurite, halo area, or percentage of degenerated axons (4,5,6)—can be subjective, are time-consuming, and are impractical for high-throughput drug screening.

Our focus into neurodegenerative disease began with the finding that activation of SIRT1 by resveratrol was linked to neuroprotection (7). Subsequently, several laboratories showed that application of exogenous nicotinamide adenine dinucleotide (NAD) protected axons from degradation (6,7). Araki and colleagues demonstrated that NAD's protective effect was circumvented by SIRT1 RNAi; however, the precise mechanism(s) of protection has not been conclusively elucidated (5,6,7,8). Having identified many new and potent SIRT1 activators in high-throughput in vitro enzyme assays, we wanted to develop the capacity to rapidly evaluate these compounds in a cell-based assay for neuroprotection. As the best of several systems tested, we chose the dorsal root ganglion (DRG) model to assess neuroprotection by NAD. In this set of experiments, we performed a dose-response experiment with varying concentrations of NAD over 90 h and scored neurodegeneration using either the previously described remaining axons or, as we have termed it, “intact neurites” (IN) method (7,8), the neurite quality index (NQI), or Neurosight (for high-throughput analysis) from images collected at different time points.

We elaborate on the details of each of these new methods herein and discuss their potential advantages. Furthermore, we propose the use of a new metric for the quantification of neuroprotection based on novel biological insight revealed by these new methods.

Monitoring neuronal demise after transection of the cell bodies from radiating neurites in a DRG explant culture is a classic model used for the study of neurodegenerative processes. The typical morphological changes that neurites undergo following trauma or toxic exposure are known as Wallerian degeneration (9). This process begins with a latent period following the trauma or toxic insult during which time no morphological changes can be detected and then progresses with swelling of the axolemma and disintegration of the cytoskeleton, forming bead-like elements along the nerve fibers (10,11). These fibers gradually fragment and become more irregular until only remnants remain (Figure 1A). Selected images from our neurodegeneration assay are shown (Figure 1B), highlighting these changes. Historically, several methods have been developed to assess Wallerian degeneration, including the counting of intact neurites and the measurement of neurite halos. For our purpose, we wanted the ability to benchmark our assays against one of these established methods and, because of its simplicity, chose IN, a binary criterion that has been widely used to quantify neurodegeneration (7,8).

Figure 1. Wallerian degeneration. (Click to enlarge)

Materials and methods

Generation of DRG explant cultures (day 1)

A single dose-response assay was performed to compare three different analytical methods to quantify neuro-protection afforded by treatment with NAD. Six DRG cultures were initially selected for each concentration of NAD to be evaluated, for the cut vehicle control and for the un-transected (uncut) vehicle control. Following culture, treatment, and transection, three to six DRGs were photographed and analyzed at each time point for each treatment group. Experimental conditions were based on the protocol utilized by Araki et al. (7). Neurites obtained by this method are mostly axons as shown by positive immunostaining for tau and negative map2 stain (data not shown) (12).

Each well of the 24-well culture plates was pre-coated with collagen (C7661; Sigma, St Louis, MO, USA) and filled with 1 mL of pre-warmed Neurobasal medium (Cat. no 21103049; Invitrogen, Carlsbad, CA, USA) freshly supplemented with B27 supplement (Cat. no. 17504–044, Invitrogen), penicillin-streptomycin (Cat. no. 15070–063, Invitrogen), and 50 µg/mL mouse nerve growth factor (mNGF, 2.5S, Cat. no. 11362348001; Roche, India-napolis, IN, USA). DRGs were collected from six CD1, E13.5 mouse embryos (Charles River Laboratories, Wilmington, MA, USA) and placed individually in pre-coated wells of the culture plates. Each DRG was centered within the well using a slightly bent 23-gauge needle on a 1-mL syringe barrel, and cultures were incubated at 37°C with 5% CO₂. The DRG cultures were allowed to mature for 12 days, during which interval one-half of the culture medium was replaced every third day with freshly supplemented medium. On day 12, fields were selected for observation using a Zeiss Axiovert 200 microscope (Carl Zeiss Microim-aging, Inc., Thornwood, NY, USA) with a mechanized stage and Axiovision software (Carl Zeiss Microimaging, Inc.). After recalling specific orientation calibration settings for each plate, the 20× objective with phase-contrast was used to identify and mark (by saving x and y coordinates) at least four distinct fields around each DRG at approximately equal distances from the center of the culture.