Neuronal cell loss is the key functional event in all neurodegenerative disorders, with apoptosis and necrosis being central to both acute and chronic degenerative processes.17
We report here, for the first time in vivo
, a technique that enables temporal resolution and quantification of the early and late phases of apoptosis and necrosis of single nerve cells using different models of disease.
The methodology permits the tracking of changes in the same cell in the same eye over hours, days, weeks and months in acute, chronic and treated glaucoma and Alzheimer models of neurodegeneration. It has enabled us to identify a numerical preponderance of late-phase versus
early-phase apoptotic cells in vivo
, and reinforced the commonalities between disease mechanisms in the different models studied. The predominance of late-phase apoptosis may reflect not only the relatively short duration of the early phase compared with the late phase (previously estimated as within 1
hour ex vivo
and suggesting that the relative likelihood of detecting cells in early apoptosis is low at any given time19
) but also, as we show here (‘similar profiles' in ), the coincidental appearance of annexin V and PI positivity in many apoptotic cells.
The shared characteristics of cell death patterns in different disease models is an important aspect of the technique presented here, with particular relevance to the design of more effective and targeted therapeutics. NMDA antagonists have long been advocated in neuroprotective strategies20, 21
in both AD22
and glaucoma,9, 21
and MK801 effectively inhibited both apoptosis and necrosis in our different models, consistent with the role of Ca2+
in mediating both forms of cell death. However, these findings support the use of our technique to investigate more specific anti-apoptotic and anti-necrotic strategies with well-defined targets, with potentially greater clinical application. It may be that the use of these strategies will depend on the stage of the disease in the patient, which may eventually be defined by an apoptotic/necrotic index, a ratio that may indicate disease severity and progression.
A major advantage of our approach is that it allows a temporal delineation of the natural history of neuronal loss. Various attempts to discriminate between the various modes of cell death in neurodegenerative diseases such as AD have been inconclusive, with mixed features of apoptosis, necrosis and autophagy being detected through a variety of histological and biochemical strategies, which are difficult to extrapolate to the dynamic events that occur in vivo
The overlap between apoptosis and necrosis pathways has been linked to insult exposure,24
with necrosis being associated with excessive pathological neuronal loss,25
now considered to be the ‘default' cell death pathway when apoptotic cell death is inhibited.26
Further investigation to elucidate whether the mode of neurodegenerative cell death is directly determined by the strength of the cell death stimulus is needed.
On the whole, most glaucoma studies have focused on the development of generic RGC loss rather than on the specific mode of cell death, although some have proposed apoptosis as the predominant pathway.27, 28, 29
Our results lead us to suggest that, although apoptosis may be involved in the primary phase of degeneration in animal models, necrosis is probably the major cell death pathway in secondary degeneration30
in glaucoma. It will be interesting to use the imaging technique developed in this study, in combination with the many fluorescent cell-permeant probes available for caspase activation, Ca2+
fluxes, mitochondrial function and so on, to examine degeneration phases in other transgenic neurodegeneration models, as the balance between apoptosis and necrosis is still unclear in chronic disease. Furthermore, delineation of the timing of such events and their association with RGC apoptosis will highlight processes that precede the irreversible stages of cellular degeneration, and provide improved and specific targets for neuroprotection.
Annexin 5 is already used systemically and safely in patients,31, 32
and there are a number of necrosis markers that are already being used in clinical trials, such as 99mTc-pyrophosphate, 111In-antimyosin and 99mTc-glucarate.32
Hence, although it is unlikely that PI can be used in patients, there are alternative agents that are potentially available to allow this methodology to be translated directly to the clinic. The ability to differentiate between apoptosis and necrosis and non-invasively track cell death in neurodegenerative pathologies could have an important role in medical diagnostics and therapy management. Furthermore, we believe that the concept of an early apoptotis/necrosis ratio with a correlation to disease activity may provide an invaluable tool for staging neurodegeneration. There is increasing evidence of the potential reversibility of early apoptosis, suggesting that a high early apoptosis/necrosis ratio may offer a good prognosis for treatment.33, 34
In conclusion, the application of this new technology has far-reaching implications for studying intracellular processes at the single-cell level in vivo. Although the equipment we use in these studies has been customised to suit animal models, the instruments are essentially the same as those used in hospitals and clinics around the world. This raises the possibility that in the near future, clinicians may be able to assess retinal nerve cell death in vivo as a method of monitoring disease progression and treatment efficacy. Whether a single snapshot or long-term in vivo observation over many weeks is needed, the ability to visualise and track changes in cell viability offers the potential for major advances in the diagnosis and management of neurodegenerative diseases.