Effects on Neurons and Hippocampal Slices by Single and Multiple Primary Blast Pressure Waves From Detonating Spherical Cyclotrimethylenetrinitramine (RDX) Explosive Charges.

Threshold shock-impulse levels required to induce cellular injury and cumulative effects upon single and/or multiple exposures are not well characterized. Currently, there are few in vitro experimental models with blast pressure waves generated by using real explosives in the laboratory for investigating the effects of primary blast-induced traumatic brain injury. An in vitro indoor experimental platform is developed using real military explosive charges to accurately represent battlefield blast exposure and to probe the effects of primary explosive blast on dissociated neurons and tissue slices. Preliminary results indicate that physical insults altered membrane permeability, impacted cellular viability, created axonal beadings, and led to synaptic protein loss in hippocampal slice cultures. Injuries from blast under the conditions that were examined did not appear to cause immediate or sustained damage to the cells. Three consecutive primary blasts failed to disrupt the overall cellular integrity in the hippocampal slice cultures and produced a unique type of pathology comprised with distinct reduction in synaptic proteins before cellular deterioration set in. These observed changes might add to the challenges in regard to enhancing our understanding of the complex biochemical and molecular mechanisms caused by primary blast-induced injury.

Blast waves from detonated military explosive reduce GluR1 and synaptophysin levels in hippocampal slice cultures.

Explosives create shockwaves that cause blast-induced neurotrauma, one of the most common types of traumatic brain injury (TBI) linked to military service. Blast-induced TBIs are often associated with reduced cognitive and behavioral functions due to a variety of factors. To study the direct effects of military explosive blasts on brain tissue, we removed systemic factors by utilizing rat hippocampal slice cultures. The long-term slice cultures were briefly sealed air-tight in serum-free medium, lowered into a 37°C water-filled tank, and small 1.7-gram assemblies of cyclotrimethylene trinitramine (RDX) were detonated 15cm outside the tank, creating a distinct shockwave recorded at the culture plate position. Compared to control mock-treated groups of slices that received equal submerge time, 1-3 blast impacts caused a dose-dependent reduction in the AMPA receptor subunit GluR1. While only a small reduction was found in hippocampal slices exposed to a single RDX blast and harvested 1-2days later, slices that received two consecutive RDX blasts 4min apart exhibited a 26-40% reduction in GluR1, and the receptor subunit was further reduced by 64-72% after three consecutive blasts. Such loss correlated with increased levels of HDAC2, a histone deacetylase implicated in stress-induced reduction of glutamatergic transmission. No evidence of synaptic marker recovery was found at 72h post-blast. The presynaptic marker synaptophysin was found to have similar susceptibility as GluR1 to the multiple explosive detonations. In contrast to the synaptic protein reductions, actin levels were unchanged, spectrin breakdown was not detected, and Fluoro-Jade B staining found no indication of degenerating neurons in slices exposed to three RDX blasts, suggesting that small, sub-lethal explosives are capable of producing selective alterations to synaptic integrity. Together, these results indicate that blast waves from military explosive cause signs of synaptic compromise without producing severe neurodegeneration, perhaps explaining the cognitive and behavioral changes in those blast-induced TBI sufferers that have no detectable neuropathology.

Effects of acute and sub-chronic L-dopa therapy on striatal L-dopa methylation and dopamine oxidation in an MPTP mouse model of Parkinsons disease.

AIMS: The molecular mechanisms for the loss of 3,4-dihydroxyphenylalanine (l-dopa) efficacy during the treatment of Parkinson's disease (PD) are unknown. Modifications related to catecholamine metabolism such as changes in l-dopa and dopamine (DA) metabolism, the modulation of catecholamine enzymes and the production of interfering metabolites are the primary concerns of this study. MAIN METHODS: Normal (saline) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) pre-treated mice were primed with 100mg/kg of l-dopa twice a day for 14 days, and a matching group remained l-dopa naïve. l-dopa naive and primed mice received a challenge dose of 100mg/kg of l-dopa and were sacrificed 30 min later. Striatal catecholamine levels and the expression and activity of catechol-O-methyltransferase (COMT) were determined. KEY FINDINGS: Normal and MPTP pre-treated animals metabolize l-dopa and DA similarly during l-dopa therapy. Administration of a challenge dose of l-dopa increased l-dopa and DA metabolism in l-dopa naïve animals, and this effect was enhanced in l-dopa primed mice. The levels of 3-OMD in MPTP pre-treated animals were almost identical to those in normal mice, which we found are likely due to increased COMT activity in MPTP pre-treated mice. SIGNIFICANCE: The results of this comparative study provide evidence that sub-chronic administration of l-dopa decreases the ability of the striatum to accumulate l-dopa and DA, due to increased metabolism via methylation and oxidation. This data supports evidence for the metabolic adaptation of the catecholamine pathway during long-term treatment with l-dopa, which may explain the causes for the loss of l-dopa efficacy.

Prenatal exposure to methanol as a dopamine system sensitization model in C57BL/6J mice.

AIMS: In this study, the effects of prenatal exposure to methanol (MeOH) on the nigrostriatal dopamine (NSDA) system were examined to determine if the interaction could sensitize this system, and serve as an underpinning for Parkinson's disease (PD) like changes that occur later in life. Methanol was studied because its toxicity resembles the symptoms of PD and the symptoms are relieved by L-dopa meaning that MeOH targets the NSDA system. Since fermentation and wood combustion are major sources for MeOH, the incidence of human encounters with MeOH is high. As a superior solvent and the precursor for formaldehyde, MeOH has a powerful and sometimes, irreversible impact on chemical processes, such as cross-linking proteins and nucleic acids. It may cause subthreshold changes that sensitizes the NSDA system to PD, that occur during aging. MAIN METHODS: To study the prenatal effects of MeOH, pregnant C57BL/6J mice were administered 40 mg/kg MeOH by oral gavage during gestation days 8-12, twice daily. Twelve weeks after birth, behavior impairments were recorded. The striatum was dissected for the determination of tyrosine hydroxylase (TH), L-aromatic amino acid decarboxylase (LAAD), α-synuclein and levels of dopamine (DA) and its metabolites. KEY FINDINGS: MeOH reduced striatal TH and LAAD protein by 47% and 57% respectively and DA by 32%. SIGNIFICANCE: The results mean that in utero exposure to toxins similar to MeOH could sensitize the striatal system to changes that cause PD. This study may help identify strategies to block this type of in utero toxicity.

L-Dihydroxyphenylalanine modulates the steady-state expression of mouse striatal tyrosine hydroxylase, aromatic L-amino acid decarboxylase, dopamine and its metabolites in an MPTP mouse model of Parkinson's disease.

AIMS: l-3,4-Dihydroxyphenylalanine (L-DOPA) is the most effective symptomatic treatment for Parkinson's disease (PD), but PD patients usually experience a successful response to L-DOPA therapy followed by a progressive loss of response. L-DOPA efficacy relies on its decarboxylation by aromatic l-amino acid decarboxylase (AAAD) to form dopamine (DA). So exogenous L-DOPA drives the reaction and AAAD becomes the rate limiting enzyme in the supply of DA. In turn, exogenous L-DOPA regulates the expression and activity of AAAD as well as the synthesis of DA and its metabolites, changes that may be linked to the efficacy and side-effects of L-DOPA. MAIN METHODS: One-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse PD model was utilized to study the effects of L-DOPA on the steady-state level and activity of AAAD, tyrosine hydroxylase (TH), DA and the metabolites of DA. The MPTP and control mice were treated twice daily with PBS or with 100mg/kg of L-DOPA for 14days and the expression and activity of AAAD, the expression of TH and the levels of DA and its metabolites were determined 24h after L-DOPA or PBS treatment, when exogenous L-DOPA is eliminated. KEY FINDINGS: In the MPTP model, L-DOPA reduced the steady-state expression and the activity of striatal AAAD by 52% and 50%, respectively, DA and metabolites were also significantly decreased. SIGNIFICANCE: The outcome shows that while L-DOPA replenishes striatal DA it also down-regulates AAAD and the steady-state synthesis and metabolic capability of the dopaminergic system. These findings are important in the precipitation of L-DOPA induced side effects and the management of L-DOPA therapy.