The contribution of initial concussive forces and resulting acrolein surge to ß-amyloid accumulation and functional alterations in neuronal networks using a TBI-on-a-chip model.

Trauma-induced Alzheimer's disease (AD) is rapidly emerging as a major consequence of traumatic brain injuries (TBI), with devastating social and economic impacts. Unfortunately, few treatment options are currently available due to a limited understanding of the underlying mechanisms. A clinically-relevant, in vitro experimental model that emulates in vivo scenarios with high levels of spatial and temporal resolution is critical for demystifying the pathways of post-TBI AD. Using a unique, recently established "TBI-on-a-chip" system with murine cortical networks, we demonstrate the correlative elevation of oxidative stress (acrolein), inflammation (TNF-α), and Aß42 aggregation, with concomitant reduction of neuronal network electrical activity post-concussive impact. These findings confirm that TBI-on-a-chip could provide a novel paradigm to supplement in vivo studies of trauma, while simultaneously validating the interaction of these alleged, key-pathological factors in post-TBI AD development. Specifically, we have shown that acrolein, acting as a diffusive factor of secondary injury, is both critical and sufficient in promoting inflammation (TNF-α) and Aß42 aggregation, two known contributors of AD pathogenesis. Furthermore, using a cell-free preparation with TBI-on-a-chip, we have confirmed that both force and acrolein can independently and directly stimulate the aggregation of purified Aß42, highlighting the key capabilities of primary and secondary injury mechanisms towards inducing Aß42 aggregation, independently and synergistically. In addition to morphological and biochemical assessment, we also demonstrate parallel monitoring of neuronal network activity, further validating the chief pathological role of acrolein in not only inflicting biochemical abnormalities, but also functional deficits in neuronal networks. In conclusion, through this line of investigations, we have shown that by recapitulating clinically-relevant events, the TBI-on-a-chip device is capable of quantitatively characterizing parallel force-dependent increases in oxidative stress, inflammation, protein aggregation, and network activity, offering a unique platform for mechanistic investigations of post-TBI AD, and trauma-induced neuronal injury in general. It is expected that this model could provide crucial insights into pathological mechanisms which will be critical in developing novel, effective diagnostics and treatment strategies that significantly benefit TBI victims.

Utilizing novel TBI-on-a-chip device to link physical impacts to neurodegeneration and decipher primary and secondary injury mechanisms.

While clinical observations have confirmed a link between the development of neurodegenerative diseases and traumatic brain injuries (TBI), there are currently no treatments available and the underlying mechanisms remain elusive. In response, we have developed an in vitro pendulum trauma model capable of imparting rapid acceleration injuries to neuronal networks grown on microelectrode arrays within a clinically relevant range of g forces, with real-time electrophysiological and morphological monitoring. By coupling a primary physical insult with the quantification of post-impact levels of known biochemical pathological markers, we demonstrate the capability of our system to delineate and investigate the primary and secondary injury mechanisms leading to post-impact neurodegeneration. Specifically, impact experiments reveal significant, force-dependent increases in the pro-inflammatory, oxidative stress marker acrolein at 24 h post-impact. The elevation of acrolein was augmented by escalating g force exposures (30-200 g), increasing the number of rapidly repeated impacts (4-6 s interval, 3, 5 and 10×), and by exposing impacted cells to 40 mM ethanol, a known comorbidity of TBI. The elevated levels of acrolein following multiple impacts could be reduced by increasing time-intervals between repeated hits. In addition, we show that conditioned media from maximally-impacted cultures can cause cellular acrolein elevation when introduced to non-impact, control networks, further solidifying acrolein's role as a diffusive-factor in post-TBI secondary injuries. Finally, morphological data reveals post-impact acrolein generation to be primarily confined to soma, with some emergence in cellular processes. In conclusion, this novel technology provides accurate, physical insults with a unique level of structural and temporal resolution, facilitating the investigation of post-TBI neurodegeneration.

Acrolein scavenger dimercaprol offers neuroprotection in an animal model of Parkinson's disease: implication of acrolein and TRPA1.

BACKGROUND: The mechanisms underlying lesions of dopaminergic (DA) neurons, an essential pathology of Parkinson's disease (PD), are largely unknown, although oxidative stress is recognized as a key factor. We have previously shown that the pro-oxidative aldehyde acrolein is a critical factor in PD pathology, and that acrolein scavenger hydralazine can reduce the elevated acrolein, mitigate DA neuron death, and alleviate motor deficits in a 6-hydroxydopamine (6-OHDA) rat model. As such, we hypothesize that a structurally distinct acrolein scavenger, dimercaprol (DP), can also offer neuroprotection and behavioral benefits. METHODS: DP was used to lower the elevated levels of acrolein in the basal ganglia of 6-OHDA rats. The acrolein levels and related pathologies were measured by immunohistochemistry. Locomotor and behavioral effects of 6-OHDA injections and DP treatment were examined using the open field test and rotarod test. Pain was assessed using mechanical allodynia, cold hypersensitivity, and plantar tests. Finally, the effects of DP were assessed in vitro on SK-N-SH dopaminergic cells exposed to acrolein. RESULTS: DP reduced acrolein and reversed the upregulation of pain-sensing transient receptor potential ankyrin 1 (TRPA1) channels in the substantia nigra, striatum, and cortex. DP also mitigated both motor and sensory deficits typical of PD. In addition, DP lowered acrolein and protected DA-like cells in vitro. Acrolein's ability to upregulate TRPA1 was also verified in vitro using cell lines. CONCLUSIONS: These results further elucidated the acrolein-mediated pathogenesis and reinforced the critical role of acrolein in PD while providing strong arguments for anti-acrolein treatments as a novel and feasible strategy to combat neurodegeneration in PD. Considering the extensive involvement of acrolein in various nervous system illnesses and beyond, anti-acrolein strategies may have wide applications and broad impacts on human health.

Simultaneous electrophysiological and morphological assessment of functional damage to neural networks in vitro after 30-300 g impacts.

An enigma of mild traumatic brain injury are observations of substantial behavior and performance deficits in the absence of bleeding or other observable structural damage. Altered behavior and performance reflect changes in action potential (AP) patterns within neuronal networks, which could result from subtle subcellular responses that affect synaptic efficacy and AP production. The aim of this study was to investigate and quantify network activity changes after simulated concussions in vitro and therewith develop a platform for simultaneous and direct observations of morphological and electrophysiological changes in neural networks. We used spontaneously active networks grown on microelectrode arrays (MEAs) to allow long-term multisite monitoring with simultaneous optical observations before and after impacts delivered by a ballistic pendulum (30 to 300 g accelerations). The monitoring of AP waveshape templates for long periods before and after impact provided an internal control for cell death or loss of cell-electrode coupling in the observed set of neurons. Network activity patterns were linked in real-time to high power phase contrast microscopy. There was no overt loss of glial or neuronal adhesion, even at high-g impacts. All recording experiments showed repeatable spike production responses: a loss of activity with recovery to near reference in 1 hr, followed by a slow activity decay to a stable, level plateau approximately 30-40% below reference. The initial recovery occurred in two steps: a rapid return of activity to an average 24% below reference, forming a level plateau lasting from 5 to 20 min, followed by a climb to within 10% of reference where a second plateau was established for 1 to 2 hrs. Cross correlation profiles revealed changes in firing hierarchy as well as in Phase 1 in spontaneous network oscillations that were reduced by as much as 20% 6-8 min post impact with only a partial recovery at 30 min. We also observed that normally stable nuclei developed irregular rotational motion after impact in 27 out of 30 networks. The evolution of network activity deficits and recovery can be linked with microscopically observable changes in the very cells that are generating the activity. The repeatable electrophysiological impact response profiles and oscillation changes can provide a quantitative basis for systematic evaluations of pharmacological intervention strategies. Future expansion to include fluorescent microscopy should allow detailed investigations of damage mechanisms on the subcellular level.

The Association of Iron and the Pathologies of Parkinson's Diseases in MPTP/MPP+-Induced Neuronal Degeneration in Non-human Primates and in Cell Culture.

Despite much efforts in the last few decades, the mechanism of degeneration of dopamine (DA) neurons in the substantia nigra (SN) in Parkinson's disease (PD) remains unclear. This represents a major knowledge gap in idiopathic and genetic forms of PD. Among various possible key factors postulated, iron metabolism has been widely suggested to be involved with fueling oxidative stress, a known factor in the pathogenesis of PD. However, the correlation between iron and DA neuron loss, specifically in the SN, has not been described in experimental animal models with great detail, with most studies utilizing rodents and, rarely, non-human primates. In the present study, aiming to gain further evidence of a pathological role of iron in PD, we have examined the correlation of iron with DA neuron loss in a non-human primate model of PD induced by MPTP. We report a significant iron accumulation accompanied by both DA degeneration in the SN and motor deficits in the monkey that displayed the most severe PD pathology and behavioral deficits. The other two monkeys subjected to MPTP displayed less severe PD pathologies and motor deficits, however, their SN iron levels were significantly lower than controls. These findings suggest that high iron may indicate and contribute to heightened MPP+-induced PD pathology in late or severe stages of PD, while depressed levels of iron may signal an early stage of disease. Similarly, using a cell culture preparation, we have found that high doses of ferric ammonium citrate (FAC), a factor known to enhance iron accumulation, increased MPP+-induced cell death in U251 and SH-SY5Y cells, and even in control cells. However, at low dose FAC restored or increased the viability of U251 and SH-SY5Y cells in the absence or presence of MPP+. These observations imply that high levels of iron likely contribute to or heighten MPP+ toxicity in the later stages of PD. While we report reduced iron levels in the earlier stages of MPTP induced PD, the significance of these changes remains to be determined.

Acrolein-mediated alpha-synuclein pathology involvement in the early post-injury pathogenesis of mild blast-induced Parkinsonian neurodegeneration.

Survivors of blast-induced traumatic brain injury (bTBI) have increased susceptibility to Parkinson's disease (PD), characterized by α-synuclein aggregation and the progressive degeneration of nigrostriatal dopaminergic neurons. Using an established bTBI rat model, we evaluated the changes of α-synuclein and tyrosine hydroxylase (TH), known hallmarks of PD, and acrolein, a reactive aldehyde and marker of oxidative stress, with the aim of revealing key pathways leading to PD post-bTBI. Indicated in both animal models of PD and TBI, acrolein is likely a point of pathogenic convergence. Here we show that after a single mild bTBI, acrolein is elevated up to a week, systemically in urine, and in whole brain tissue, specifically the substantia nigra and striatum. Acrolein elevation is accompanied by heightened α-synuclein oligomerization, dopaminergic dysregulation, and acrolein/α-synuclein interaction in the same brain regions. We further show that acrolein can directly modify and oligomerize α-synuclein in vitro. Taken together, our data suggests acrolein likely plays an important role in inducing PD pathology following bTBI by encouraging α-synuclein aggregation. These results are expected to advance our understanding of the long-term post-bTBI pathological changes leading to the development of PD, and suggest intervention targets to curtail such pathology.