The current results extend the preliminary results reported by Petrik et al. [8
] by showing that microglial activation is part of the underlying pathology in the lumbar cord. These data add to those previously reported, i.e., the loss of motor and other neurons and the activation of reactive astrocytes. Taken together with the current data, the overall activation of a glial inflammatory response in lumbar cord suggests that this process is a key early stage of the pathological events leading to motor neuron death. This interpretation is supported by an absence of motor neuron loss and astrocyte activation in the other levels of the spinal cord observed in the present study. In ALS and in animal models of the disease, glial activation followed by motor neuron death often appears to proceed in sequential manner along the ventral neuraxis with the first signs of pathology appearing first in lumbar cord [31
]. Given this, it seems possible that an examination of later time points would show pathological responses in the thoracic and cervical cord as well. Alternatively, the aluminum shown to be present in lumbar cord motor neurons may not have reached these other spinal cord segments. Studies now in progress will determine if motor neurons in these other segments stain positively for aluminum.
The positive Morin staining in lumbar cord clearly demonstrates that post injection aluminum finds entry into this part of the nervous system. One possibility is that it does so by retrograde transport from muscles to motor neurons in particular segments. This seems unlikely given that our paradigm of injecting subcutaneous should not have targeted any particular spinal cord segment. Another possibility is that aluminum can enter the CNS in a systemic manner if it enters the circulatory system. Experiments in progress are designed to distinguish between these possibilities.
The presence of hyper-phosphorylated tau protein, one of the hallmarks of both Alzheimer's disease and ALS–PDC of Guam, in motor neurons in lumbar spinal cord clearly suggests that additional pathological processes associated with aluminum are occurring.
The behavioural outcomes in the second experiment reported here reinforce the pathological outcomes seen in the first studies. While the histological measurements from these studies are still pending, the extent of the behavioural deficits strongly suggests that we will observe widespread neuronal pathologies. The greater extent of the behavioural outcomes in this experiment may be related to the experimental paradigm that tripled the number of aluminum hydroxide injections.
Overall, the results reported here mirror previous work that has clearly demonstrated that aluminum, in both oral and injected forms, can be neurotoxic [15
]. Potential toxic mechanisms of action for aluminum may include enhancement of inflammation (i.e., microgliosis) and the interference with cholinergic projections [34
], reduced glucose utilization [33
], defective phosphorylation-dephosphorylation reactions [35
], altered rate of transmembrane diffusion and selective changes in saturable transport systems in the blood brain barrier (BBB [36
], and oxidative damage on cellular processes by the inhibition of the glutathione redox cycle [37
Given the above, it is not surprising that aluminum has been widely proposed as a factor in neurodegenerative diseases and has been found in association with degenerating neurons in specific CNS regions [38
]. In animal studies, aluminum has been linked to the accumulation of tau protein and amyloid-beta protein and observed to induce neuronal apoptosis in vivo
as well as in vitro30
. Aluminum injected animals show severe anterograde degeneration of cholinergic terminals in cortex and hippocampus [42
Aluminum in its adjuvant form can gain access to the CNS [42
], however, oral administration of aluminum hydroxide gel does not appear to be neurotoxic in humans [45
], although aluminum chloride is, in rats [46
]. The route of exposure, and perhaps the form of aluminum, may be important factors that determine the potential for toxicity.
We speculate that the observed neurotoxic effects of aluminum hydroxide in the present study arise by both ‘direct’ and ‘indirect’ pathways, some of which are cited above. Direct toxicity refers to the physical presence (or close proximity) of aluminum and its potential for initiating cell death pathways. Accumulation of aluminum into the cytoplasm via cellular uptake mechanisms or diffusion could cause alterations in glutaminase and glutamine synthetase and easily alter the availability of the neurotransmitter glutamate [47
]. Aluminum acting to induce abnormal tau protein accumulation could also increase neurofibrillary tangles and impair cellular transport mechanisms [48
]. Outside the cell, aluminum could affect neurons by altering synapses. For example, aluminum has been shown to decrease the thickness of post-synaptic density, increase the width of the synaptic cleft, and increase the number of flat synapses [49
]. Aluminum could also block voltage-activated calcium channels [50
], augment the activity of acetylcholinesterase [51
], or interfere with synaptic transmission by merely accumulating in the synaptic cleft [52
]. Aluminum can also induce apoptosis in astrocytes [53
]. Since astrocytes are essential for maintaining neuronal health, any loss of astrocyte function could prove toxic to neurons. Indirect toxicity of aluminum could occur in various ways, including by activating various cytokines [54
], releasing glutamate in an excitotoxic cascade, or by modifying various enzymatic pathways [55
In addition to the above actions specifically on neural cells, aluminum might act indirectly by stimulating abnormal, generalized immune responses. This is, in fact, what adjuvants are placed in vaccines to do in the first place. Adjuvant neurotoxicity could thus be the result of an imbalanced immune response. Rook and Zumla [56
] hypothesized that multiple vaccinations, stress, and the method of vaccination could lead to a shift in immune response [56
]. Aluminum hydroxide has previously been shown to stimulate a Th2-cytokine response [9
While the current results and our previous study have demonstrated significant behavioural and neuropathological outcomes with aluminum hydroxide and some additionally significant outcomes due to a combination of adjuvants, it is important to recognize that these were achieved under minimal
conditions. summarizes aspects of human ALS and GWS symptoms compared with outcomes observed in aluminum-injected mice. The likelihood exists that a synergistic effect between adjuvants and other variables such as stress, multiple vaccinations, and exposure to other toxins likely occurs. A recent study examining some of these factors in combination showed that stress, vaccination, and pyridostigmine bromide (a carbamate anticholinesterase (AchE) inhibitor), may synergistically act on multiples stress-activated kinases in the brain to induce neurological impairments in GWS [59
]. In addition, a genetic background in context to aluminum exposure may play a crucial role and may be an important area for future research.
The demonstration of neuropathological outcomes and behavioural deficits in aluminum hydroxide injected mice may provide some insight into the causes of not only GWS–ALS, but may open avenues of investigation into other neurological diseases.