Gulf War illness (GWI) affects a significant percentage of veterans of the 1991 conflict, but its origin remains unknown. Associated with some cases of GWI are increased incidences of amyotrophic lateral sclerosis and other neurological disorders. Whereas many environmental factors have been linked to GWI, the role of the anthrax vaccine has come under increasing scrutiny. Among the vaccine’s potentially toxic components are the adjuvants aluminum hydroxide and squalene. To examine whether these compounds might contribute to neuronal deficits associated with GWI, an animal model for examining the potential neurological impact of aluminum hydroxide, squalene, or aluminum hydroxide combined with squalene was developed. Young, male colony CD-1 mice were injected with the adjuvants at doses equivalent to those given to US military service personnel. All mice were subjected to a battery of motor and cognitive-behavioral tests over a 6-mo period postinjections. Following sacrifice, central nervous system tissues were examined using immunohistochemistry for evidence of inflammation and cell death. Behavioral testing showed motor deficits in the aluminum treatment group that expressed as a progressive decrease in strength measured by the wire-mesh hang test (final deficit at 24 wk; about 50%). Significant cognitive deficits in water-maze learning were observed in the combined aluminum and squalene group (4.3 errors per trial) compared with the controls (0.2 errors per trial) after 20 wk. Apoptotic neurons were identified in aluminum-injected animals that showed significantly increased activated caspase-3 labeling in lumbar spinal cord (255%) and primary motor cortex (192%) compared with the controls. Aluminum-treated groups also showed significant motor neuron loss (35%) and increased numbers of astrocytes (350%) in the lumbar spinal cord. The findings suggest a possible role for the aluminum adjuvant in some neurological features associated with GWI and possibly an additional role for the combination of adjuvants.
Abou-Donia M. B., Wilmarth K. R., Jensen K. F., Oehme F. W., and Kurt T. L. (1996) Neurotoxicity resulting from coexposure to pyridostigmine bromide, deet, and permethrin: implications of Gulf War chemical exposures. J. Toxicol. Environ. Health 48, 35–56.
Asa P. B., Cao, Y., and Garry R. F. (2000) Antibodies to squalene in Gulf War syndrome. Exp. Mol. Pathol. 68, 55–64.
Asa P. B., Wilson R. B., and Garry R. F. (2002) Antibodies to squalene in recipients of anthrax vacine. Exp. Mol. Pathol. 73, 19–27.
Banks W. A. and Kastin A. J. (1989) Aluminum-induced neurotoxicity: alterations in membrane function at the blood-brain barrier. Neurosci. Biobehav. Rev. 13, 47–53.
Barbeito L. H., Pehar M., Cassina P., et al. (2004) A role for astrocytes in motor neuron loss in amyotrophic lateral sclerosis. Brain Res. Brain Res. Rev. 47, 263–274.
Baylor N. W., Egan W., and Richman P. (2002) Aluminum salts in vaccines—US perspective. Vaccine 20(Suppl 3), S18-S23.
Benisek Z., Suli, J., Elias D., et al. (2004) Experimental squalene adjuvant. II. Harmlessness and local reactogenity. Vaccine 22, 3470–3474.
Bilkei-Gorzo A. (1993) Neurotoxic effect of enteral aluminium. Food Chem. Toxicol. 31, 357–361.
Brewer J. M., Conacher M., Hunter C. A., Mohrs M., Brombacher F., and Alexander J. (1999) Aluminium hydroxide adjuvant initiates strong antigen-specific Th2 responses in the absence of IL-4-or IL-13-mediated signaling. J. Immunol. 163, 6448–6454.
Bromberg M. B. (2002) Diagnostic criteria and outcome measurement of amyotrophic lateral sclerosis. Adv. Neurol. 88, 53–62.
Carlson B. C., Jansson A. M., Larsson A., Bucht A., and Lorentzen J. C. (2000) The endogenous adjuvant squalene can induce a chronic T-cell-mediated arthritis in rats. Am. J. Pathol. 156, 2057–2065.
Charatan F. (2002) US links motor neurone disease with Gulf war service. BMJ 324, 65.
Crapper D. R., Krishnan S. S., and Dalton A. J. (1973) Brain aluminum distribution in Alzheimer’s disease and experimental neurofibrillary degeneration. Science 180, 511–513.
Crawley J. N. (2000) What’s Wrong With My Mouse? Behavioral Phenotyping of Trangenic and Knockout Mice. 65–69.
Crawley J. N., Belknap J. K., Collins A., et al. (1997) Behavioral phenotypes of inbred mouse strains: implications and recommendations for molecular studies. Psychopharmacology (Berl.) 132, 107–124.
DeFries, J. C., Hegmann, J. P., and Halcomb, R. A. (1974) Response to 20 generations of selection for open-field activity in mice. Behav. Biol. 11, 481–495.
Duan W. R., Garner D. S., Williams, S. D., Funckes-Shippy C. L., Spath I. S., and Blomme E. A. (2003) Comparison of immunohistochemistry for activated caspase-3 and cleaved cytokeratin 18 with the TUNEL method for quantification of apoptosis in histological sections of PC-3 subcutaneous xenografts. J. Pathol. 199, 221–228.
Dyer O. (2004) Inquiry finds that Gulf war veterans face extra burden of disease. BMJ 329, 1257.
Eisen A. and Weber M. (2001) The motor cortex and amyotrophic lateral sclerosis. Muscle Nerve 24, 564–573.
Everts H. G. and Koolhaas J. M. (1999) Differential modulation of lateral septal vasopressin receptor blockade in spatial learning, social recognition, and anxiety-related behaviors in rats. Behav. Brain Res. 99, 7–16.
Fellows P. F., Linscott M. K., Ivins B. E., et al. (2001) Efficacy of a human anthrax vaccine in guinea pigs, rabbits, and rhesus macaques against challenge by Bacillus anthracis isolates of diverse geographical origin. Vaccine 19, 3241–3247.
Ferguson E. and Cassaday H. J. (2001 and 2002) Theoretical accounts of Gulf War Syndrome: from environmental toxins to psychoneuroimmunology and neurodegeneration. Behav. Neurol. 13, 133–147.
Lick-Smith H. C., Waters E. L., Walker N. J., et al. (2005) Mouse model characterisation for anthrax vaccine development: comparison of one inbred and one outbred mouse strain. Microb. Pathol. 38, 33–40.
Fukuda K., Nisenbaum R., Stewart G., et al. (1998) Chronic multisymptom illness affecting Air Force veterans of the Gulf War. JAMA 280, 981–988.
Fulco C. E., Liverman C. T., and Sox H. C. (2000) Gulf War and Health: Volume 1. Depleted Uranium, Pyridostigmine, Bromide, Sarin, and Vaccines. Institute of Medicine. National Academy Press, pp. 89–168.
Gabutti G., Guido M., Durando P., et al. (2005) Safety and immunogenicity of conventional subunit and MF59-adjuvanted influenza vaccines in human immunodeficiency virus-1-seropositive patients. J. Int. Med. Res. 33, 406–416.
Gajkowska B., Smialek M., Ostrowski R. P., Piotrowski P., and Frontczak-Baniewicz M. (1999) The experimental squalence encephaloneuropathy in the rat. Exp. Toxicol. Pathol. 51, 75–80.
Garruto R. M., Shankar S. K., Yanagihara R., Salazar A. M., Amyx H. L., and Gajdusek D. C. (1989) Low-calcium, high-aluminum diet-induced motor neuron pathology in cynomolgus monkeys. Acta Neuropathol. (Berl) 78, 210–219.
Haley R. W. (2003) Excess incidence of ALS in young Gulf War veterans. Neurology 61, 750–756.
Haley R. W., Kurt T. L., and Hom J. (1997) Is there a Gulf War Syndrome? Searching for syndromes by factor analysis of symptoms. JAMA. 277, 215–222.
Hom J., Haley R. W., and Kurt T. L. (1997) Neuropsychological correlates of Gulf War syndrome. Arch Clin. Neuropsychol. 12, 531–544.
Horner R. D., Kamins K. G., Feussner J. R., et al. (2003) Occurrence of amyotrophic lateral sclerosis among Gulf War Veterans. Neurology 61, 742–749.
Hotopf M., David A., Hull, L., Ismail K., Unwin C., and Wessely S. (2000) Role of vaccinations as risk factors for ill health in veterans of the Gulf war: cross sectional study. BMJ 320, 1363–1367.
Hunter D., Zoutman D., Whitehead J., Hutchings J., and MacDonald K. (2004) Health effects of anthrax vaccination in the Canadian forces. Mil Med. 169, 833–838.
Ivins B., Fellows P., Pitt L., et al. (1995) Experimental anthrax vaccines: efficacy of adjuvants combined with protective antigen against an aerosol Bacillus anthracis spore challenge in guinea pigs. Vaccine 13, 1779–1784.
Jefferson T., Rudin M., and Di Pietrantonj C. (2004) Adverse events after immunisation with aluminium-containing DTP vaccines: systematic review of the evidence. Lancet Infect Dis 4, 84–90.
Joshi J. G. (1990) Aluminum, a neurotoxin which affects diverse metabolic reactions. Biofactors 2, 163–169.
Kalra R., Singh S. P., Razani-Boroujerdi S., et al. (2002) Subclinical doses of the nerve gas sarin impair T cell responses through the autonomic nervous system. Toxicol. Appl. Pharmacol. 184, 82–87.
Kang H. K., Mahan C. M. Lee K. Y., et al. (2002) Evidence for a deployment-related GulfWar syndrome by factor analysis. Arch. Environ. Health 57, 61–68.
Kanra G., Viviani S., Yurdakok K., et al. (2003) Effect of aluminum adjuvants on safety and immunogenicity of Haemophilus influenzae typeb-CRM197 conjugate vaccine. Pediatr. Int. 45, 314–318.
Kawahara M., Kato M., and Kuroda Y. (2001) Effects of aluminum on the neurotoxicity of primary cultured neurons and on the aggregation of betaamyloid protein. Brain Res. Bull. 55, 211–217.
Kurland L. T. (1988) Amyotrophic lateral sclerosis and Parkinson’s disease complex on Guam linked to an environmental neurotoxin. Trends Neurosci. 11, 51–54.
Kurt T. L. (1998) Epidemiological association in US veterans between Gulf War illness and exposures to anticholinesterases. Toxicol. Lett. 102–103, 523–536.
Lee V. M., Page C. D., Wu H. L., and Schlaepfer W. W. (1984) Monoclonalantibodies to gel-excised glial filament protein and their reactivities with other intermediate filament proteins. J. Neurochem. 42, 25–32.
Levine, J. B., Kong J., Nadler M., and Xu Z. (1999) Astrocytes interact intimately with degenerating motor neurons in mouse amyotrophic lateral sclerosis (ALS). Glia 28, 215–224.
Maatkamp A., Vlug A., Haasdijk E., Troost D., French P. J., and Jaarsma D. (2004) Decrease of Hsp25 protein expression precedes degeneration of motoneurons in ALS-SOD1 mice. Eur. J. Neurosci. 20, 14–28.
Matyas G. R., Rao M., Pittman P. R., et al. (2004) Detection of antibodies to squalene: III. Naturally occurring antibodies to squalene in humans and mice. J. Immunol. Methods 286, 47–67.
Morris R. (1984) Developments of a water-maze procedure for studying spatial learning in the rat. J. Neurosci. Methods 11, 47–60.
Mullen R. J., Buck C. R., and Smith A. M. (1992) Neu N, a neuronal specific nuclear protein in vertebrates. Development 116, 201–211.
Murakami N. (1999) Parkinsonism-dementia complex on Guam—overview of clinical aspects. J. Neurol. 246(Suppl. 2), II16-II18.
Nagy D., Kato T., and Kushner P. D. (1994) Reactive astrocytes are widespread in the cortical gray matter of amyotrophic lateral sclerosis. J. Neurosci. Res. 38, 336–347.
Nass M. (1999) Anthrax vaccine. Model of a response to the biologic warfare threat. Infect Dis. Clin. North Am. 13, VIII187-VIII208.
Nass M. (2002) The Anthrax Vaccine Program: an analysis of the CDC’s recommendations for vaccine use. Am. J. Public Health 92, 715–721.
Nass M., Fisher B. L., and Robinson S. (2005) Comments and Questions regarding FDA’s proposed rule and order to licnese Anthrax Vaccine Absorbed. FDA Anthrax vaccine docket submission. Proposed rule and proposed order. 29 Fed. Reg. 78, 281–78, 293. December 29, 2004.
Nicolson G. L., Nasralla M. Y., Haier J., and Pomfret J. (2002) High frequency of systemic mycoplasmal infections in Gulf War veterans and civilians with Amyotrophic Lateral Sclerosis (ALS). J. Clin. Neurosci. 9, 525–529.
O’Reilly S. A., Roedica J., Nagy D., et al. (1995) Motor neuron-astrocyte interactions and levels of Cu, Zn superoxide dismutase in sporadic amyotrophic lateral sclerosis. Exp. Neurol. 131, 203–210.
Paxinos G. and Franklin K. B. J. (2001) The Mouse Brain in Stereotoxic Coordinates 2nd ed. Academic Press. Sydney.
Perl D. P., Gajdusek D. C., Garruto R. M., Yanagihara R. T., and Gibbs C. J. (1982) Intraneuronal aluminum accumulation in amyotrophic lateral sclerosis and Parkinsonism-dementia of Guam. Science, 217, 1053–1055.
Perl D. P. and Pendlebury W. W. (1986) Aluminum neurotoxicity—potential role in the pathogenesis of neurofibrillary tangle formation. Can. J. Neurol. Sci. 13, 441–445.
Plaisier M. (2000) Letter dated March 20, 2000 from Department of Health and Human Services to former US member of Congress, Rep. Jack Metcalf, admitting to squalene, in anthrax vaccine while denying that it was in the licencsed formulation.
Platt B., Fiddler G., Riedel G., and Henderson Z. (2001) Aluminium toxicity in the rat brain: histochemical and immunocytochemical evidence. Brain Res. Bull. 55, 257–267.
Rao J. K., Katsetos C. D., Herman M. M., and Savory J. (1998) Experimental aluminum ecephalomyelopathy. Relationship to human neurodegenerative disease. Clin. Lab. Med. 18, VIII687-VIII698.
Redhead K., Quinlan G. J., Das R. G., and Gutteridge J. M. (1992) Aluminium-adjuvanted vaccines transiently increase aluminium levels in murine brain tissue. Pharmacol. Toxicol. 70, 278–280.
Rook G. A. and Zumla A. (1997) Gulf War syndrome: is it due to a systemic shift in cytokine balance towards a Th2 profile?. Lancet 349, 1831–1833.
Rook G. A. and Zumla A. (1998) Is the Gulf War syndrome an immunologically mediated phenomenon? Hosp. Med. 59, 10–11.
Sahin G., Varol I., Temizer A., Benli K., Demirdamar R., and Duru S. (1994) Determination of aluminum levels in the kidney, liver, and brain of mice treated with aluminum hydroxide. Biol. Trace Elem. Res. 41, 129–135.
Salamon R., Verret C., Jutand M. A., et al. (2006) Health consequences of the first Persian Gulf War on French troops. Int J Epidemiol. 35, 479–487.
Samson K. (2002) VA study finds ALS spike in Gulf War vets. Neurol. Today 2(1), 13–14.
Sartin J. S. (2000) Gulf War illnesses: causes and controversies. Mayo Clin. Proc. 75, 811–819.
Sasaki S., Tsutsumi Y., Yamane K., Sakuma H., and Maruyama S. (1992) Sporadic amyotrophic lateral sclerosis with extensive neurological involvement. Acta Neuropathol. (Berl.) 84, 211–215.
Satoh M., Kuroda Y., Yoshida H., et al. (2003) Induction of lupus autoantibodies by adjuvants. J. Autoimmun, 21, 1–9.
Savory J. and Garruto R. M. (1998) Aluminum, tau protein, and Alzheimer’s disease: an important link? Nutrition 14, 313–314.
Schumm W. R., Webb F. J., Jurich A. P., and Bollman S. R. (2002a) Comments on the Institute of Medicine’s 2002 report on the safety of anthrax vaccine. Psychol. Rep. 91, 187–191.
Schumm W. R., Reppert E. J., Jurich A. P., et al. (2002b) Self-reported changes in subjective health and anthrax vaccination as reported by over 900 Persian Gulf War era veterans. Psychol. Rep. 90, 639–653.
Schumm W. R., Jurich A. P., Bollman S. R., Webb F. J., and Castelo C. S. (2005) The long term safety of anthrax vaccine, pyridostigmine bromide (PB) tablets, and other risk factors among Reserve Component Veterans of the First Persian Gulf War. Medical Veritas 2, 348–362.
Shaw C. A. and Wilson J. M. (2003) Analysis of neurological disease in four dimensions: insight from ALS-PDC epidemiology and animal models. Neurosci. Biobehav. Rev. 27, 493–505.
Shawky S. (2002) Depleted uranium: an overview of its properties and health effects. East Mediterr. Health J. 8, 432–439.
Sidman R. L., Angevine J. B. Jr., and Pierce E. T. (1971) Atlas of the Mouse Brain and Spinal Cord.
Skowera A., Hotopf M., Sawicka E., et al. (2004) Cellular immune activation in Gulf War veterans. J. Clin. Immunol. 24, 66–73.
Steele L. (2000) Prevalence and patterns of Gulf War illness in Kansas veterans: association of symptoms with characteristics of person, place, and time of military service. Am. J. Epidemiol. 152, 992–1002.
Suli J., Benisek Z., Elias D., et al. (2004) Experimental squalene adjuvant. I. Preparation and testing of its effectiveness. Vaccine 22, 3464–3469.
Taylor D. N., Sanchez J. L., Smoak B. L., and DeFraites R. (1997) Helicobacter pylori infection in Desert Storm troops. Clin. Infect Dis. 25, 979–982.
Tohyama T., Lee V. M., Rorke L. B., and Trojanowski J. Q. (1991) Molecular milestones that signal axonal maturation and the commitment of human spinal cord precursor cells to the neuronal or glial phenotype in development. J. Comp. Neurol. 310, 285–299.
Tsuchiya K., Takahashi M., Shiotsu H., et al. (2002) Sporadic amyotrophic lateral sclerosis with circumscribed temporal atrophy: a report of an autopsy case without dementia and with ubiquitinated intraneuronal inclusions. Neuropathology 22, 308–316.
Unwin C., Blatchley N., Coker W., et al. (1999) Health of UK servicemen who served in Persian Gulf War. Lancet 353, 169–178.
Valensi J. P., Carlson J. R., and Van Nest G. A. (1994) Systemic cytokine profiles in BALB/c mice immunized with trivalent influenza vaccine containing MF59 oil emulsion and other advanced adjuvants. J. Immunol. 153, 4029–4039.
Wagner-Recio M., Toews A. D., and Morell P. (1991) Tellurium blocks cholesterol synthesis by inhibiting squalene metabolism: preferential vulnerability to this metabolic block leads to peripheral nervous system demyelination. J. Neurochem. 57, 1891–1901.
Wang D., Perides G., and Liu Y. F. (2005) Vaccination alone or in combination with pyridostigmine promotes and prolongs activation of stress-activated kinases induced by stress in the mouse brain. J. Neurochem. 93, 1010–1020.
Weisskopf M. G., O’Reilly E. J., McCullough M. L., et al. (2005) Prospective study of military service and mortality from ALS. Neurology 64, 32–37.
Wen G. Y. and Wisniewski H. M. (1985) Histochemical localization of aluminum in the rabbit CNS. Acta Neuropathol (Berl.) 68, 175–184.
Wetts R. and Vaughn J. E. (1996) Differential vulnerability of two subsets of spinal motor neurons in amyotrophic lateral sclerosis. Exp. Neurol. 141, 248–255.
Williamson E. D., Hodgson I., Walker N. J., et al. (2005) Immunogenicity of recombinant protective antigen and efficacy against aerosol challenge with anthrax. Infect Immun. 73, 5978–5987
Wolf H. K., Buslei R., Schmidt-Kastner R., et al. (1996) NeuN: a useful neuronal marker for diagnostic histopathology. J. Histochem. Cytochem. 44, 1167–1171
Wolfe J., Proctor S. P., Erickson D. J., and Hu H. (2002) Risk factors for multisymptom illness in US Army veterans of the Gulf War. J. Occup. Environ. Med. 44, 271–281.
About this article
Cite this article
Petrik, M.S., Wong, M.C., Tabata, R.C. et al. Aluminum adjuvant linked to gulf war illness induces motor neuron death in mice. Neuromol Med 9, 83–100 (2007). https://doi.org/10.1385/NMM:9:1:83
- Amyotrophic Lateral Sclerosis
- Motor Neuron
- Aluminum Hydroxide
- Lumbar Spinal Cord