SOCIETA' ITALIANA DI FARMACOLOGIA
Relazioni dei borsisti sif
RELAZIONE
ATTIVITA’ SCIENTIFICA all'ESTERO
Dott.ssa Irene Paterniti
Dipartimento Clinico-Sperimentale di Medicina e Farmacologia
Università degli Studi di Messina
The main objective of the project, entitled “Can omega-3 fatty acids represent a new therapeutic approach in the treatment of spinal cord trauma?", was to investigate the neuroprotective and neurotrophic properties of omega-3 polyunsaturated fatty acids (PUFA), and in particular docosahexaenoic acid (DHA), as a potential treatment of traumatic neuronal injury.
In the first phase of this project we evaluated the anti-inflammatory and anti-oxidant effects of omega-3 fatty acids, in an experimental model of spinal cord injury (SCI) that causes traumatic damage in the central nervous system (CNS).
SCI was induced in the mouse by application of aneurysm clips (force of 24 g), to replicate the persistence of cord compression that is commonly observed in human SCI.
Thirty minutes after compression, animals received a tail vein injection of DHA at a dose of 500nmol/kg and all animals were killed 24 h after SCI in order to evaluate various parameters implicated in the development of injury.
The results of this first phase of the project showed that DHA improves the key parameters involved in inflammation, axonal destruction and demyelination at the site of impact and in the perilesional area, confirming the neuroprotective effects of DHA after spinal cord trauma.
During SCI neurons within both the peripheral nervous system (PNS) and the CNS can be damaged, resulting in pain and loss of function.
Injury to the spinal cord arises from many types of trauma; however, this initial trauma often leads to two principal forces being exerted onto the nervous tissue, that in turn lead to injury and cell death. These forces are compression and distraction, and SCI commonly involves the combination, in varying degrees, of both of these forces.
Following a primary mechanical insult there is a cascade of downstream events, termed secondary injury, which occurs hours to days after the initial event, and include: microvascular dysfunction at the site of injury, recruitment and activation of inflammatory cells associated with secretion of cytokines, which contribute to further tissue damage, and cellular apoptosis.
The second purpose of this project was to develop an in vitro injury model that could replicate some of the events that follow traumatic neuronal injuryin vivo.
Primary cultures of dorsal root ganglion (DRG) neurons from rat and mouse have been studied for many years, in the laboratory of Prof. Adina Michael-Titus and Prof. John Priestley at the Blizard Institute in the Centre for Neuroscience and Trauma Queen Mary University of London, and others worldwide, and have therefore been well characterized. SCI is known to affect the dorsal root and its ganglions, and, although these neurons are essentially located in the PNS, their axons form central connections with the dorsal horn of the spinal cord.
DRG cells are sensory neurons that transmit mechanoreceptive, thermoreceptive and nociceptive information from the periphery to the spinal cord. For many years, DRG cells have been characterized based on their anatomy and physiology, but in recent years, neurochemical studies have revealed a remarkable number of additional subpopulations.
In addition, the DRG phenotype is relatively plastic, changing in response to nerve and (or) tissue damage and generating further heterogeneity.
In this study we decided to examine the neurotoxic effects of the oxidative stress induced by the hydrogen peroxide (H2O2), to mimic some of the secondary injury pathways that characterize SCI, and assess how the neuroprotective properties of DHA (as previously described) could increase the survival of cultured DRG neurons following exposure to H2O2 .
Oxidative stress induces cell injury in a number of neuronal cell culture models and by a variety of proposed mechanisms. Reactive oxygen species (ROS), which include superoxide anion, hydroxyl radical and hydrogen peroxide, cause DNA damage by directly attacking DNA or by activation of endonucleases that degrade DNA, and ultimately contribute to cell death.
In this study, primary neuronal cultures were prepared according to the method of Gavazzi and colleagues (1999), with some modifications. Cervical to lumbar level DRGs were removed and cleaned before being dissociated chemically with 0.125% collagenase at 37°C for 45 minutes.
DRG neurons were cultured on laminin-coated glass coverslips for 48 h before being subjected to H2O2 (10nM) and DHA at final concentration of 1 microM for 24 h.
Exposure of DRG cultured cells to H2O2 resulted in a disruption of neurites, decrease in neurite outgrowth, and a decrease in the number of cells.
Cultures treated with DHA (1 microM) appeared to have less disruption of neurites (the neurites had the best growth in the DHA-treated cultures) and an increase in the number of cells with outgrowths compared with H2O2-treated cells.
In conclusion, the in vitro models discussed above allowed for a further investigation of the neuroprotective effects of omega-3 fatty acids in adult DRG cultures.
The ability to protect neurons is crucial to a successful recovery after SCI, since regeneration can only occur from neurons that survive the initial trauma.
This work demonstrated that DHA was neuroprotective when given as a post-injury treatment. This suggests that DHA could still be of benefit when administered after a traumatic injury, which is an important factor when considering translation to the clinic.
Signed : 16 December 2011
Professor Adina Michael-Titus - Professor of Neuroscience
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