ARTICLE |
Correspondence to: Lee J. Martin, Johns Hopkins Univ. School of Medicine, Dept. of Pathology, 558 Ross Building, 720 Rutland Ave., Baltimore, MD 21205-2196. E-mail: lmartin@jhmi.edu
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We developed an isolation technique for motor neurons from adult rat spinal cord. Spinal cord enlargements were discretely microdissected into ventral horn tissue columns that were trypsin-digested and subjected to differential low-speed centrifugation to fractionate ventral horn cell types. A fraction enriched in -motor neurons was isolated. Motor neuron enrichment was verified by immunofluorescence for choline acetyltransferase and prelabeling axon projections to skeletal muscle. Adult motor neurons were isolated from naïve rats and were exposed to oxidative agents or were isolated from rats with sciatic nerve lesions (avulsions). We tested the hypothesis, using single-cell gel electrophoresis (comet assay), that hydrogen peroxide, nitric oxide, and peroxynitrite exposure in vitro and axotomy in vivo induce DNA damage in adult motor neurons early during their degeneration. This study contributes three important developments in the study of motor neurons. It demonstrates that mature spinal motor neurons can be isolated and used for in vitro models of motor neuron degeneration. It shows that adult motor neurons can be isolated from in vivo models of motor neuron degeneration and evaluated on a single-cell basis. This study also demonstrates that the comet assay is a feasible method for measuring DNA damage in individual motor neurons. Using these methods, we conclude that motor neurons undergoing oxidative stress from reactive oxygen species and axotomy accumulate DNA damage early in their degeneration. (J Histochem Cytochem 49:957972, 2001)
Key Words: amyotrophic lateral sclerosis, apoptosis, axotomy, DNA single-strand breaks, single-cell gel electrophoresis
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
GENOTOXICITY, caused by DNA damage, is believed to contribute to the mechanisms of human aging and disease (
A variety of endogenous or intrinsic and exogenous or environmental factors can cause DNA damage (10,000 lesions per day due to metabolism-generated free radicals (
-rays, free radicals, and heavy metals. These exogenous agents can cause crosslinks, adducts, and oxidative cleavage. Many of these types of DNA damage can be converted from one form to another form. For example, the interaction of DNA with hydroxyl radicals yields a variety of lesions, including base adducts, strand breaks, and sugar modifications; in addition, AP sites are converted into SSBs if not repaired.
DNA lesions can be evaluated by a variety of methods. Some methods are quantitative and other methods are qualitative. The methods for DNA damage detection can be broadly classified as biochemical and in situ detection methods. DNA filter elution assay is a powerful biochemical method for the determination of DNA strand breaks, apurinic/apyrimidinic (alkali-labile) sites, and crosslinks (
We are interested in developing sensitive and quantitative assays for detecting genotoxicity directly in motor neurons because of its relevance to ALS (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals and Tissues
Adult male SpragueDawley rats (Charles River; Wilmington, MA) weighing 150200 g were used for these experiments. Naïve rats without experimental manipulations and rats with experimental manipulations were used in these experiments. The manipulations performed were retrograde tracing of motor neurons and avulsion of the sciatic nerve. The institutional Animal Care and Use Committee approved the animal protocols. The animals were housed in a colony room with a 12 hr:12 hr light:dark cycle and ad libitum access to food and water.
Retrograde Tracing of Spinal Motor Neurons
Rats (n=2) were anesthetized deeply with enflurane:oxygen:nitrous oxide (1:33:66) and, using sterile surgery, the sciatic nerve was exposed within the middle of the upper hindlimb. The nerve was transected by cutting and 50 µl of 2.5% DAPI was applied to the proximal nerve stump for 1 hr using tracer-saturated Gelfoam placed in a sterile Eppendorf tube. The incision was closed with the bottom of the Eppendorf tube containing DAPI remaining applied to the proximal stump of the sciatic nerve. The animals were allowed to survive for 48 hr before they were sacrificed.
Sciatic Nerve Avulsion Model of Motor Neuron Apoptosis
The unilateral sciatic nerve avulsion model was used as an in vivo model of spinal motor neuron apoptosis (
Preparation of Spinal Cord Tissue for Isolation of Mature Motor Neurons
Spinal cords were isolated from adult rats that were anesthetized deeply with a mixture of enflurane:oxygen:nitrous oxide (1:33:66) and then decapitated. From rats without experimental lesions, cervical and lumbar enlargements were used. The two enlargements of the spinal cord contain the majority of the spinal motor neurons. From rats exposed to tracers and from rats with sciatic nerve avulsions, only the entire lumbar enlargements (divided into ipsilateral and contralateral sides) were used. After removal of the pia, lumbar/cervical enlargements were dissected segmentally under a surgical microscope and then the segments were microdissected into gray matter columns of ventral horn without appreciable contamination of dorsal horn and surrounding white matter funiculi. Spinal motor neurons are large neurons locating in Lamina IX of spinal cord. Gray matter tissue columns from spinal cord ventral horns of lumbar/cervical enlargements were collected and rinsed in a cell culture dish on ice containing dissection medium [1 x Ca2+ and Mg2+-free Hanks balanced salt solution (Gibco BRL; Grand Island, NY) supplemented with glucose and sucrose]. These tissues were used to prepare motor neuron cell suspensions.
Preparation of Adult Spinal Motor Neuron-enriched Cell Suspensions
Digestion of Spinal Cord Ventral Horns.
Ventral horn samples were digested (20 min) with 0.25% trypsinEDTA (Gibco) in a tissue culture incubator (5% CO2 and 95% air at 37C). This mixture was titurated gently with a transfer pipette. The tissue digested was transferred to a 5-ml centrifugation tube on ice, and the remaining small pieces of ventral horn gray matter were further digested in trypsinEDTA (16 min). The total cell suspension was then centrifuged at different speeds for cell sorting.
Sorting of Cell Suspensions
To isolate a spinal motor neuron-enriched fraction, tissue digests were centrifuged (Beckman GPR model centrifuge) at 200 rpm (20 gav) for 5 min (4C). The supernatant was collected and then centrifuged at 400 rpm (50 gav), 800 rpm (160 gav), and then 2500 rpm (1400 gav). After each spin (for 5 min), the pellet was resuspended in 100 µl PBS, pH 7.4, fixed with 1 ml of 4% paraformaldehyde (4C for 1 hr) for cell characterization using immunocytochemistry, TUNEL, or cresyl violet staining.
Characterization of Sorted Cell Suspensions
The cells were repelleted after fixation and each pellet was resuspended with 250 µl PBS. An aliquot of cell suspension (50 µl) was applied to a gelatin-coated slide and a coverslip (24 mm x 30 mm) was gently overlaid to form a monolayer of cells. The slides were then air-dried. Air-dried slides were rinsed (1 hr) in PBS to separate the slides from the coverslips. The cells did not attach to the coverslips because they were not coated with adhesive; instead, the cells were attached to the gelatin-coated slides. The cells were permeabilized (30 min) in 1% Triton X-100 and then treated (30 min) with 1% bovine serum albumin (BSA). The cells were probed with antibodies to neuronal nucleus protein (NeuN, diluted 1:20), a neuron-specific marker (Chemicon International; Temecula, CA), choline acetyltransferase (ChAT, diluted 1:5), a marker for motor neurons in rat spinal cord enlargements (Roche Molecular Biochemicals; Indianapolis, IN), glial fibrillary acidic protein (GFAP, diluted 1:20), an astroglial marker (DAKO; Glostrup, Denmark), CD11b/c IgG2a (OX-42, diluted 1:20), a microglial/macrophage marker (Harlan Sera-Lab; Sussex, UK). Diluted primary antibodies were applied to the slides and the slides were incubated (24 hr at room temperature) in a humidified box. After primary antibody incubation, the slides were rinsed in PBS. Alexa-conjugated anti-mouse IgG (diluted 1:100; Molecular Probes, Eugene, OR) was used to visualize NeuN, ChAT, and OX-42, and Cascade blue-conjugated anti-rabbit IgG (diluted 1:100; Molecular Probes) was used to visualize GFAP. The slides were incubated (4 hr at RT) in a humidified dark box. The slides were washed and coverslipped with propidium iodide/antifade (Ventana; Tucson, Arizona). The slides were observed and photographed under a Zeiss fluorescence microscope. The preparation of cell suspensions from rats used for retrograde tracing of motor neurons was identical to that described above. The slides were coverslipped with or without propidium iodide/antifade and were observed under the same fluorescence microscope but with UV emission.
Counting of Different Cell Types in Cell Suspensions
To identify the cell fraction that was enriched in motor neurons, neurons marked with cell-specific antibodies were counted. The total number of neurons was estimated by comparing the number of NeuN-positive cells to the total number of cells identified by propidium iodide staining plus NeuN staining. To determine the proportion of spinal motor neurons in the cell suspensions, the percentage of ChAT-positive cells relative to NeuN-positive cells was calculated. To determine the proportion of spinal motor neurons issuing sciatic nerve axons, the fraction of DAPI-positive cells relative to the total number of NeuN-positive cells was also calculated. The numbers of labeled cells from six different microscopic fields (x400) were averaged from each case and then a total mean was derived from the preparations from three different cases.
Immunoblotting
Immunoblotting was used to identify ChAT immunoreactivity in lysates of motor neuron cell suspensions to additionally verify the presence of a motor neuron phenotype in this fraction. The cells were pelleted, washed, and lysed in buffer. Samples (20 µg of total protein) were fractionated by SDS-PAGE. Proteins were electroeluted onto nitrocellulose sheets. Blots were washed with 50 mM Tris-buffered saline (TBS) and blocked in 2.5% nonfat milk in 50 mM TBS/0.1% Tween-20. ChAT immunoreactivity was detected with a monoclonal antibody (Incstar, Stillwell, MN) used at a concentration of 1 µg IgG/ml. Immunoreactivity was visualized with enhanced chemiluminesence.
In Vitro Exposure of Motor Neurons to ROS
We tested the hypothesis that adult motor neurons rapidly accumulate genomic DNA lesions in response to oxidative stress. Motor neuron-enriched cell suspensions were exposed to H2O2, NO donors, H2O2 + NO donor, and ONOO-. Two different NO donors were used: sodium nitroprusside (SNP; Sigma, St Louis, MO) and N-(2-aminoethyl)-N-(2-hydroxyl-nitrosohydrazino)-1,2-ethylenediamine (spermine-NONOate; OXIS International, Portland, OR). SpermineNONOate was used because this agent can maintain long exposure to steady-state generation of NO (
Motor neuron cell suspensions (the 400 rpm preparation) were prepared from naïve rats. Motor neurons were exposed to SNP at concentrations of 10, 100, and 300 µM for different durations ranging from 15 min to 4 hr. Identical cell suspensions were exposed to spermineNONOate at concentrations of 10 and 100 µM for 30 min, 1 hr, and 2 hr. Alternatively, motor neurons were treated with ONOO- at concentrations of 10 and 100 µM for 15 min, 30 min, and 1 hr. These exposures were done in medium containing 90% Neurobasal-A (Gibco), 5% horse serum, 5% fetal bovine serum (both sera were heat-inactivated) and 1 x glutamine (Gibco) in a tissue culture incubator (containing 5% CO2 and 95% air, 37C) for the different times. For controls, samples of the same cell suspensions were incubated in medium for the same time in the absence of SNP, with spermine tetrahydrochloride/sodium nitrite (NO2-) or with decomposed ONOO- in alkaline solution. After exposure, the treatment groups were collected in 5-ml centrifuge tubes and repelleted at 4C for 5 min. Each pellet was resuspended and subjected to the comet assay.
Comet Assay
Preparation of Cell Microgels on Slides.
To detect DNA damage in individual cells, motor neuron cell suspensions that were exposed to H2O2, NO donor, H2O2/SNP, and ONOO- were analyzed by the comet assay. In addition, to identify DNA damage in motor neurons undergoing apoptosis in vivo, the comet assay was used on motor neuron cell suspensions prepared from rats with sciatic nerve avulsions. The 400 rpm cell preparations from ipsilateral or contralateral sides of ventral horns of lumbar enlargements of animals with unilateral sciatic nerve avulsions were subjected directly to comet assay immediately after they were sorted and repelleted. All the procedures for comet assay were done under low light to minimize spontaneous DNA damage.
Our method for the comet assay on motor neurons is based mainly on the original protocol for lymphocytes (4.4 x 104 motor neurons) and low melting-point agarose was applied to the first gel layer. The slides were then coverslipped and placed at 4C for solidification of the cell suspensionagarose mixture. After the second layer solidified, the coverslips were removed and 100 µl of low melting-point agarose was added on top of the cell layer. The gels were re-coverslipped and the slides were placed on ice for gel solidification.
For preparing microgels, we compared two brands of low melting-point agarose (at the same concentrations) from different companies: Gibco BRL LMP agarose (cat. no. 15517-022) and FMC BioProducts SeaPlaque GTG agarose (cat. no. 50110). We found that the FMC agarose was the best for our purpose.
Lysis of Cells, DNA Unwinding, Gel Electrophoresis, and DNA Staining.
Coverslips were removed from the cell microgels and the slides were covered with 1.5 ml of lysis buffer at pH 10 (for alkaline conditions) or pH 8.6 (for neutralized conditions) containing 2.5 M NaCl, 100 mM EDTA, 1% sodium lauryl sarcosine, 10 mM Tris, and Triton X-100 (final concentration 1%, freshly added immediately before use). The cell microgels were lysed for 30 min (at RT). After draining, microgels were treated with DNA-unwinding solution (300 mM NaOH, 1 mM EDTA, generally at pH 12 unless otherwise stated) for 30 min at RT. In some experiments, the effects of DNA unwinding solution/electrophoresis buffer pH on comet patterns were studied. Three different pH conditions were used: pH 13, pH 12, and pH 7.4. Loss of a purine or pyrimidine base from the DNA sugarphosphate backbone facilitates an alkali-catalyzed ß-elimination of the 3'-phosphate (
13, alkali-labile sites are converted to SSB (
Counting Comets in Microgels
The number of cells with comets were counted in microgels prepared from motor neurons exposed to ROS in vitro and from motor neurons isolated from in vivo axotomy experiments. For cell preparations exposed to H2O2, NO donor, H2O2/SNP or ONOO-, cells incubated for the same time in medium without oxidant were used as controls. Three to five separate experiments from different animals were done for each type of in vitro oxidant exposure experiment. For the unilateral sciatic nerve avulsion experiments, comet assays were performed on two to four rats for each recovery time (5, 7, 10, 14, or 28 days). The contralateral (unlesioned) side of the spinal cord from rats with sciatic nerve avulsions was used as the control for each time point. The number of comets and large intact cell nuclei regarded as motor neurons stained by ethidium bromide were counted in six microscopic views at x200 from microgels of treated cells and from sciatic nerve avulsion animals. The percentages of comets relative to the total number of cells (total number of comets and total number of intact cell nuclei) were determined and group means were calculated. The data were analyzed using a Student's t-test.
TUNEL
Motor neuron cell suspensions exposed to NONOate (10 µM or 100 µM) or corresponding vehicle (10 µM or 100 µM spermine/NO2-) were analyzed with TUNEL. Cells were fixed in 4% paraformaldehyde overnight. They were pelleted and washed with PBS (pH 7.4), and then mounted on gelatin-coated slides by coverslipping to form a cell monolayer and allowed to air-dry. The slides were rinsed in PBS for 1 hr to separate the coverslip from the slides. A modified enhanced TUNEL procedure was used. The cells were rinsed in 1% Triton X-100 for 1 hr and washed with PBS before they were rinsed with terminal deoxynucleotidyl transferase (TdT) buffer (containing 30 mM Tris, pH 7.2, 140 mM sodium cacodylate, 3 mM cobalt chloride) for 20 min. The TdT buffer was changed to fresh TdT buffer (0.5 ml/slide) containing TdT (0.02U/µl) and biotin-16-dUTP (50 µM) (both reagents were from Roche Molecular Biochemistry) and the slides were incubated at 37C for 2 hr. The reaction was terminated by incubating the slides in SSC (300 mM sodium chloride, 30 mM sodium citrate) for 15 min. The slides were then incubated in avidinbiotinperoxidase complex for 2 hr at RT. After washing with PBS, the slides were stained with DAB/H2O2 for 20 min. Some of the slides were counterstained with cresyl violet and were observed and photographed.
Double Labeling of Comet Assay Microgels with Immunocytochemistry
We evaluated the feasibility of characterizing motor neuron comets by immunocytochemistry. Motor neurons were examined for expression of survival motor neuron (SMN) protein and p53 in microgels after comet assay. Coverslips from microgels of motor neurons exposed to either H2O2 (for SMN) or NONOate (for p53) were removed after they were stained with ethidium bromide and observed for comets. Mouse monoclonal antibodies were used to localize SMN (Transduction Laboratories; Lexington, KY) and p53 (Santa Cruz; Santa Cruz, CA). Microgels were incubated with primary antibody dilutions (1:250 for SMN and 1:50 for p53) for 24 hr at RT. The slides were washed with PBS, then incubated (1:501:100 dilution) with Alexa-conjugated anti-mouse IgG for 2 hr at RT. Afterwards, the slides were washed and re-coverslipped for observation and photography.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
New approaches need to be developed to study mechanisms of degeneration of adult motor neurons. Specifically, sensitive quantitative assays for specific types of DNA lesions need to be identified to study the possible role of DNA damage in the upstream mechanisms of motor neuron apoptosis. We developed and characterized a novel short-term, motor neuron-enriched cell suspension system to evaluate the formation of DNA damage directly in individual adult spinal motor neurons.
Purification of a Motor Neuron-enriched Cell Suspension
Ventral horns of cervical/lumbar enlargements of adult rat spinal cord were microdissected and subjected to mild dissociation with trypsinEDTA. The total cell suspension was fractionated by low-speed centrifugation. The different cellular fractions were examined for NeuN, ChAT, GFAP, and OX-42 immunofluorescence, and for DAPI fluorescence to identify -motor neurons giving rise to sciatic nerve axons. One fraction was identified that is enriched in motor neurons. In the 400 rpm fraction of digests of ventral horn enlargements, neurons comprise
84% of the total cell number (NeuN-positive cells/total cells, i.e., NeuN-positive cells and cells stained with propidium iodide; Fig 1A). Of these neurons,
86% are motor neurons (ChAT-positive cells/NeuN-labeled cells; Fig 1B and Fig 1C). Of these motor neurons,
72% are
-motor neurons giving rise to sciatic nerve axons (by DAPI sciatic nerve tracing; Fig 1F). Some motor neurons are entirely or partially surrounded by fragments of astroglial processes (Fig 1D and Fig 1C), but GFAP-positive cells are rarely present in this fraction. OX-42-positive microglial cells are present only occasionally (not shown). Immunoblotting evaluation of fraction for ChAT confirmed the presence of motor neurons in this fraction (Fig 2).
|
|
DNA Damage Is Rapidly Induced and Accumulates in Motor Neurons Undergoing Oxidative Stress
Motor neuron cell suspensions were exposed to H2O2, NO donor, H2O2/SNP, or ONOO-. Control cells were exposed to vehicle and incubated in medium for the same time. Comet assays were done at pH 12 (at this pH hydrogen bonds in DNA destabilize, causing strand separation). Comets were observed with each treatment (Fig 3). Each treatment gave consistent results, with the major comet pattern generated from each exposure being highly reproducible (Fig 3). Fig 3 shows the most common patterns of comets after different treatments.
|
In microgels of control samples, the nuclei of many intact cells stained with ethidium bromide. Motor neurons with intact genomic DNA in gels stained with ethidium bromide have an evenly stained, smooth round nucleus without a tail (Fig 3A), indicating no DNA damage. In control motor neuron suspensions incubated in Neurobasal-A medium, the percentage of comets is 10% or less. Control comets have a large round head, densely labeled with ethidium bromide, and a short granular tail, composed of large scattered granules (Fig 3A). We have reported previously (
|
A comparison of control comets (Fig 3A) with comets induced by H2O2 (Fig 3B) and NO donors (Fig 3D3H) shows the striking difference in the comet profiles and amount (Table 1) of DNA damage. With 1 mM H2O2 exposure in neurobasal-A medium, the comets have a round head, a tail with fine granules, and a short neck between head and tail (Fig 3B). With 1 mM H2O2 exposure in MEM, the comets have huge spindle-shaped tails with a small round head (Fig 3C), indicating severe DNA damage and elution from the cell. Exposure to NO donors generates consistent comet profiles depending on exposure concentration and duration. In general, with exposure to 10 µM SNP or NONOate for 15 min, 30 min, or even 1 hr, the comet head has a halo composed of large granules, with a short granular tail (Fig 3D and Fig 3E). With 100 µM or higher doses, or with 10 µM for more than 1 hr of exposure, the head halo is less prominent but the tail length increases (Table 1), and it is still composed of scattered large granules (Fig 3F). Another comet pattern that shows severe DNA damage is found after motor neurons are exposed to 100 µM SNP with 1 mM H2O2 (Fig 3G). With these comets the head is small and the tail is long and wide; the degree of DNA damage is severe (Table 1). With increased exposure to 100 µM SNP with 1 mM H2O2, the comet head contains some clumped DNA, as shown by ethidium bromide staining (Fig 3H). We have found previously that the combination of SNP with 1 mM H2O2 induces severe protein nitration in motor neuron suspensions (
|
Measurement of DNA Damage
The comet moment is regarded as one of the best indices of induced DNA damage in cells (1030 comets in each treatment group) were captured as digital images using Inquiry software (Loats Associates; Westminster, MD). Comets were saved in TIFF format. Each comet was used to obtain several measurements by delineating the region of interest (Fig 5). DNA intensityhead, DNA intensitytail, areahead, and areatail were measured. Tail length was measured from the center of the head to the end of the tail. Relative DNA content is reflected by the average integrated intensity of fluorescence. The amount of DNA damage for each cell is derived from the calculation of the comet moment (
|
The comets in control motor neurons have low moments (1.7 x 105; Table 1). The comet moments in motor neurons exposed to ROS are significantly higher than those of controls (Table 1). These measurements show that NO, NO plus H2O2, and ONOO- induce DNA damage directly in motor neurons. In addition, the results show that the comet moment is a sensitive measure of DNA damage in motor neurons.
DNA Damage Occurs Early in Motor Neurons Undergoing Apoptosis In Vivo
We applied the comet assay to an in vivo model of motor neuron apoptosis in adult spinal cord. Sciatic nerve avulsion in adult rat causes apoptosis of lumbar motor neurons over 714 days (
Identification of Different Types of DNA Damage in Motor Neurons by Varying pH Conditions
The use of different pH conditions during electrophoresis is an approach to discriminate between DNA strand breaks and alkali-labile sites (13, alkali-labile sites are thought to be converted into SSBs, and DNA crosslinking diminishes migration of DNA strand breaks. At pH 13, we did not observe comets in motor neurons exposed to 10 mM H2O2 in neurobasal-A for several durations (30, 45, 60, and 90 min). At pH 12, many comets are observed after exposure of motor neurons to 10 mM H2O2 (
1:2000) instead of DSB or alkali-labile sites (
We evaluated the types of DNA damage (SSBs or alkali-labile sites) induced in motor neurons exposed to NO (Fig 6). At two different concentrations of NONOate (10 and 100 µM, exposure for 1 or 2 hr), the number of comets detected at pH 12 was generally greater than the number observed at pH 13. The comet morphology was different at pH 12 and, compared to pH 13 (Fig 7A and Fig 7B). At pH 12, comets had prominent halos and short tails (Fig 7A), and comets at pH 13 had no halos and long granular tails (Fig 7B). At pH conditions of 12.6 or higher, alkali-labile sites are converted to SSBs and, therefore a pH 13 maximizes the detection of alkali-labile sites as SSBs (
|
|
The types of DNA damage were analyzed in motor neurons exposed to ONOO- (Fig 8). ONOO- induces rapid formation of alkali-labile sites, followed by an accumulation of SSBs while alkali-labile sites decline (Fig 8A). In addition, ONOO- induces DSBs (pH 7.4) very quickly in motor neurons (Fig 7C and Fig 8B). These changes in motor neuron DNA were not the result of generalized cellular degradation because ChAT levels remained stable (Fig 2). Therefore, ONOO- is a potent DNA-damaging agent that concurrently induces alkali-labile sites, SSBs, and DSBs in motor neurons.
|
TUNEL Analysis of Isolated Adult Motor Neurons Exposed to NO Donor
TUNEL is a commonly used method for detecting DNA damage in cells. For comparison with the comet assay, we used the TUNEL method on motor neurons exposed to NO donor. Motor neuron cell suspensions were exposed to NONOate (10 µM or 100 µM) or corresponding vehicle (10 µM or 100 µM spermine/NO2-). TUNEL confirmed that NO is toxic to motor neurons because many motor neurons were TUNEL-positive after exposure to NONOate (Fig 9B9E) but not after exposure to spermine/NO2- (Fig 9A). TUNEL staining ranged from light (Fig 9C and Fig 9D) to very dark and aggregated (Fig 9E). However, the types of DNA damage could not be ascertained with the TUNEL method; yet, a major advantage of the TUNEL method over the comet assay is that the morphological progression of motor neuron apoptosis is better appreciated with the TUNEL method (Fig 9E).
|
Combination of Comet Assay with Immunocytochemistry
We evaluated whether comet assay and immunocytochemical techniques could be combined. This combination is desirable because information on the expression of specific proteins is helpful for understanding the mechanisms of DNA damage-induced motor neuron apoptosis (
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This work advances the study of motor neurons in three ways. It shows that mature spinal motor neurons can be isolated and used for in vitro models of neurotoxicity. This demonstration was extended by showing that adult motor neurons can also be isolated from in vivo models of motor neuron degeneration and evaluated for DNA damage using single-cell analysis. This study also shows for the first time that the comet assay is a useful method for measuring distinct DNA lesions in individual motor neurons. Using the comet assay with different pH conditions, we identified the coexistence of different types of early DNA damage in motor neurons. This approach may enable the study of motor neuron disease to move in new directions, particularly with regard to understanding mechanisms of DNA damage-induced apoptosis of motor neurons, which is relevant to ALS (
Mature Motor Neurons Can Be Studied In Vitro
We developed a new approach to study mature motor neurons by creating a short-term, motor neuron-enriched cell suspension isolated from spinal cord ventral horn enlargements of adult rat. The motor neuron enrichment of this cell system was confirmed by immunophenotyping (e.g., ChAT, NeuN, and SMN), retrograde tracing, and immunoblotting. Electron microscopy has also been used to confirm the presence of motor neurons in this preparation (
We took advantage of the structural features (i.e., their large size) of adult motor neurons and the large difference in size between spinal motor neurons and surrounding cells in the ventral horn to assist in their isolation by centrifugation. With strict microdissection and appropriate dissociation, our cell-sorting method may also be useful for isolating other populations of selectively vulnerable neurons, including cerebellar Purkinje cells, CA1 pyramidal neurons, and nigral dopaminergic neurons. This procedure could be highly applicable for Purkinje cells because these neurons are much larger than any adjacent cells, although the appropriate centrifugation speeds and times need to be identified for neuronal types other than spinal motor neurons.
This method is an important technical advancement in the field of motor neuron degeneration because very few methods are available to study motor neurons in vitro. Embryonic motor neuron cultures are a widely used in vitro system (40% survival at 24 hr after isolation;
Early DNA Damage in Motor Neurons Can Be Measured with the Comet Assay
Since Kohn discovered that single-strand DNA is eluted rapidly under the alkaline condition from cells onto filters, the alkaline elution method has been used to detect and measure low-level DNA damage in eukaryotic cells (12, DNA DSBs at neutralized conditions, DNADNA or DNAprotein crosslinking, alkali-labile sites that cause SSBs under alkali conditions (pH >12.6), and damage to purine and pyrimidine bases (AP sites). The comet assay is sensitive enough for detecting one break per 2 x 1010 Daltons of DNA in lymphocytes (
For one approach, we isolated motor neurons using our new method and exposed these cells in vitro to different ROS, followed by analysis by the comet assay. Motor neuron-enriched cell suspensions were exposed to H2O2, NO donors, H2O2 + NO donor, and ONOO-. We tested the hypothesis that oxidative stress causes adult motor neurons to accumulate DNA damage. We used different pH conditions during electrophoresis to discriminate between DNA strand breaks and alkali-labile sites in motor neurons. Interestingly, we found that different ROS induce different DNA damage signatures in neurons, which has not been shown before. H2O2 induces primarily SSBs in motor neurons, consistent with previous reports using non-neuronal cells (
In the other model we used an in vivo lesion that induces motor neuron apoptosis (
The comet assay has advantages over other more frequently used methods for detecting DNA damage (e.g., nick end-labeling methods such as TUNEL). It appears that assays for single-stranded DNA are more sensitive and specific than TUNEL for apoptosis. We have confirmed indirectly the sensitivity of comet assay detection of DNA SSBs as an early and sensitive marker for apoptosis. We found that DNA SSBs are visualized prominently in avulsed motor neurons at least 2 days before the detection of DNA fragmentation by TUNEL (
Previous studies using the comet assay have not combined this method with immunophenotyping of neuronal populations with DNA damage. We anticipated that some nuclear proteins would retain antigenicity in comet assay microgels. This suspicion was confirmed. Motor neurons contained SMN within the nucleus. In cell cultures (
![]() |
Acknowledgments |
---|
Supported by grants from the US Public Health Service, the National Institutes of Health, National Institute of Neurological Disorders and Stroke (NS34100), and National Institute on Aging (AG16282), and the Department of Defense, US Army Medical Research and Materiel Command (DAMD17-99-1-9553).
Received for publication March 12, 2001; accepted March 14, 2001.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ames BN (1989) Endogenous DNA damage as related to cancer and aging. Mutat Res 214:41-46[Medline]
Beckman JS, Carson M, Smith CD, Koppenol WH (1993) ALS, SOD and peroxynitrite. Nature 364:548[Medline]
Gavrieli Y, Sherman Y, Ben-Sasson SA (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493-501[Abstract]
Hellman B, Vaghef H, Boström B (1995) The concept of tail moment and tail inertia in the single cell gel electrophoresis assay. Mutat Res 336:123-131[Medline]
Henderson CE, BlochGallego E, Camu W (1995) Purified embryonic motoneurons. In Cohen J, Wilkin G, eds. Nerve Cell Culture: a Practical Approach. London, Oxford University Press, 69-81
Horváthová E, SlameÚová D, Gábelová A (1999) Use of single cell gel electrophoresis (comet assay) modifications for analysis of DNA damage. Gen Physiol Biophys 18:70-74[Medline]
Hrabie JA, Klose JR, Wink DA, Keefer LK (1993) New nitric oxide-releasing zwitterions derived from polyamines. J Org Chem 58:1472-1476
Jayaraman L, Prives C (1995) Activation of p53 sequence-specific DNA binding by short single strands of DNA requires the p53 C-terminus. Cell 81:1021-1029[Medline]
Kindzelskii AL, Petty HR (1999) Ultrasensitive detection of hydrogen peroxide-mediated DNA damage after alkaline single cell gel electrophoresis using occultation microscopy and TUNEL labeling. Mutat Res 426:11-22[Medline]
Kisby GE, Kabel H, Hugon J, Spencer P (1999) Damage and repair of nerve cell DNA in toxic stress. Drug Metab Rev 31:589-618[Medline]
Kohn KW (1991) Principles and practice of DNA filter elution. Pharmacol Ther 49:55-77[Medline]
Kohn KW, Erìckson LC, Ewig RAG, Friedman CA (1976) Fractionation of DNA from mammalian cells by alkaline elution. Biochemistry 15:4629-4637[Medline]
Lai H, Singh NP (1995) Acute low-intensity microwave exposure increases DNA single-strand breaks in rat brain cells. Bioelectromagnetics 16:207-210[Medline]
Levine AJ (1997) p53, the cellular gatekeeper for growth and division. Cell 88:323-331[Medline]
Lindahl T (1993) Instability and decay of the primary structure of DNA. Nature 362:709-715[Medline]
Liu Q, Dreyfuss G (1996) A novel nuclear structure containing the survival of motor neurons protein. EMBO J 15:3555-3565[Abstract]
Liu Z, Martin LJ (2001) Motor neurons rapidly accumulate DNA single strand breaks after in vitro exposure to nitric oxide and peroxynitrite and in vivo axotomy. J Comp Neurol 432:35-60[Medline]
Martin LJ (1999) Neuronal death in amyotrophic lateral sclerosis is apoptosis: possible contribution of a programmed cell death mechanism. J Neuropathol Exp Neurol 58:459-471[Medline]
Martin LJ (2000) p53 is abnormally elevated and active in the CNS of patients with amyotrophic lateral sclerosis. Neurobiol Dis 7:613-622[Medline]
Martin LJ, Kaiser A, Price AC (1999) Motor neuron degeneration after sciatic nerve avulsion in adult rat evolves with oxidative stress and is apoptosis. J Neurobiol 40:185-201[Medline]
Martin LJ, Price AC, Kaiser A, Shaikh AY, Liu Z (2000) Mechanisms for neuronal degeneration in amyotrophic lateral sclerosis and in models of motor neuron death. Int J Mol Med 5:3-13[Medline]
Morris EJ, Drexiler JC, Cheng K-Y, Wilson PM, Gin RM, Geller HM (1999) Optimization of single-cell gel electrophoresis (SCGE) for quantitative analysis of neuronal DNA damage. BioTechniques 26:282-289[Medline]
Östling O, Johanson KJ (1984) Microelectrophoretic study of radiation-induced DNA damage in individual mammalian cells. Biochem Biophys Res Commun 123:291-298[Medline]
Pagliardini S, Giavazzi A, Setola V, Lizier C, DiLuca M, DeBiasi S, Battaglia G (2000) Subcellular localization and axonal transport of the survival motor neuron (SMN) protein in the developing rat spinal cord. Hum Mol Genet 9:47-56
Petersen AB, Gniadecki R, Wulf HC (2000) Laser scanning cytometry for comet assay analysis. Cytometry 39:10-15[Medline]
PorteraCailliau C, Price DL, Martin LJ (1997) Non-NMDA and NMDA receptor-mediated excitotoxic neuronal deaths in adult brain are morphologically distinct: further evidence for an apoptosis-necrosis continuum. J Comp Neurol 378:87-104
Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184-191[Medline]
Subba Rao K (1993) Genomic damage and its repair in young and aging brain. Mol Neurobiol 7:23-48[Medline]
Tice RR, Agurell E, Anderson D, Burlison B, Hartmann A, Kobayashi H, Miyamae Y, Rojas E, Ryu J-C, Sasaki YF (2000) Single cell gel/comet assay: guidelines for in vitro and an in vivo genetic toxicological testing. Environ Mol Mutagen 35:206-221[Medline]