Improved outcome after spinal cord compression injury in mice treated with docosahexaenoic acid
Highlights
► A compression model of thoracic spinal cord injury is described in the mouse. ► Compression injury results in a long-lasting locomotor deficit and cell loss. ► DHA delivered acutely post-injury improves recovery and reduces cell loss. ► However chronic dietary DHA is not neuroprotective following compression injury.
Introduction
Traumatic spinal cord injury (SCI) has devastating consequences for patients, and current treatments, such as acute decompression (Fehlings and Perrin, 2006) or administration of methylprednisolone (Hurlbert and Hamilton, 2008), are controversial or of limited efficacy. In preclinical studies, major progress has been made in both the fields of neuroprotection and neuroregeneration (Kwon et al., 2010), but translation into the clinic has been disappointing. Neuroprotective strategies are particularly appealing because they aim to stop the spread of injury, and the complex cascade of events that follow the primary insult means there are many possible targets for neuroprotective agents. However, although there are promising new agents under development (Kwon et al., 2011a), many clinical trials have failed, either because of limited efficacy or unexpected toxicity (Tator and Fehlings, 1999). There is, therefore, still a need for the development of new neuroprotective agents, which are safe and effective when delivered after spinal cord injury.
Recently, a number of preclinical studies have demonstrated that omega-3 (n − 3) polyunsaturated fatty acids (PUFAs) are neuroprotective when administered after SCI. n − 3 PUFAs are essential fatty acids that have crucial roles in the development and mature functioning of the nervous system. Particularly important is docosahexaenoic acid (DHA), a long chain (22 carbon) n − 3 PUFA that accounts for approximately 50% of the PUFAs in central nervous system (CNS) membranes. We have shown in rat hemisection and compression models of SCI that DHA administered as an intravenous bolus 30 min after injury leads to increased neuronal, oligodendrocyte and axonal survival at the lesion epicentre, and improved locomotor function (Huang et al., 2007b, King et al., 2006, Ward et al., 2010). When the acute bolus is combined with dietary DHA supplementation for several weeks following injury, additional cell and axonal survival is seen, and further improvement of functional outcome (Huang et al., 2007b, Ward et al., 2010). Most recently we have shown that a multi-nutrient dietary formulation containing n − 3 PUFAs is also neuroprotective (Zbarski-Barquero et al., 2012). Neuroprotection has also been reported in studies involving treatment with DHA prior to SCI (Figueroa et al., 2012) or treatment after SCI with fenretinide, a synthetic retinoid derivative which increases levels of endogenous DHA (Lopez-Vales et al., 2010). DHA has also been shown to be neuroprotective if administered after traumatic brain injury in rodents (Bailes and Mills, 2010, Wu et al., 2011). The exact mechanism underlying these neuroprotective effects is not known, but it is likely to involve multiple pathways. Actions of DHA include effects on membrane properties such as fluidity and permeability (Stillwell and Wassall, 2003), effects on membrane proteins including receptors (Lafourcade et al., 2011) and ion channels (Lauritzen et al., 2000), effects on gene transcription by direct binding of non-esterified DHA to peroxisome proliferator activated receptors (PPARs) (Jump, 2002), reduction in the levels of pro-inflammatory n − 6 PUFAs by competition for metabolic and catabolic pathways, and effects of DHA metabolites, including the anti-inflammatory and pro-homeostatic neuroprotectin D1 (NPD1) (Bazan et al., 2011). These multiple actions, and the established safety profile of existing n − 3 PUFA formulations (Heller et al., 2006), make DHA a very attractive candidate for the clinical treatment of acute SCI.
Before DHA is tested in clinical trials, it should be validated in a wide range of preclinical SCI models, including different types of injury (e.g. contusion and compression) and different species (e.g. rat and mouse). Efficacy in multiple models has been identified as an important criterion before translation to the clinic (Kwon et al., 2011b). Most of our studies of n − 3 PUFAs have been carried out in a rat thoracic compression model of SCI (Hall et al., 2012, Huang et al., 2007b, Lim et al., 2010, Ward et al., 2010), a model in which cell death extends over a longer period than in contusion injury models (Huang et al., 2007a). In the present study we have therefore developed a mouse compression model, which is similar in principle to our rat model (Huang et al., 2007a) and used it to investigate the neuroprotective properties of acute and dietary DHA treatment. It is well-established that mice exhibit a very different response to SCI, both in terms of functional recovery, and also in the tissue pathological changes and inflammatory reaction, compared to that seen in other mammals. In particular, mice do not exhibit the progressive necrosis and larger central cavitation that is so dramatic in rats and other mammals, in which a rim of anatomically preserved white matter surrounds a fluid-filled cystic cavity at the injury epicentre (Grossman et al., 2001, Huang et al., 2007a). In contrast, the injured mouse spinal cord is completely filled-in with dense fibrous connective tissue, and if present, there are only very small cavities (microcysts) in the lesion site (Farooque, 2000, Joshi and Fehlings, 2002a, Joshi and Fehlings, 2002b, Ma et al., 2001). The use of a mouse compression model therefore allows us to test DHA in a very different pathology, and provides a baseline for future studies in transgenic mice (e.g. Lim et al., 2012).
Section snippets
Compression SCI
C57BL/6 female mice (19–21 g) were deeply anaesthetized with 4% isoflurane (Merial, Essex, UK), as evidenced by lack of response to a nociceptive stimulus. Subsequent anaesthesia throughout the procedure was maintained using 1.5–2% isoflurane, with oxygen and nitrous oxide at a 1:1 ratio. A laminectomy was performed at vertebral level T12, leaving the dura undisturbed. The T11 and T13 transverse processes were clamped in a spinal compression frame, and the compression was applied by suspending
Locomotor recovery after compression SCI
Compression SCI at vertebral T12 level produced a long-lasting deficit in hindlimb function, as assessed using the BMS scale (Fig. 1). Recovering from complete paralysis, all mice demonstrated plantar placement of the paw (BMS score 3) by 2 weeks after injury, and some mice started to show occasional weight-supported plantar stepping (BMS score 4). Beyond 2 weeks the mice continued to recover very slowly and by 4 weeks had plateaued at a BMS score of just above 4 (4.2). This equates with
Discussion
In this study we have characterised a novel paradigm for compression SCI in the mouse, and used it to test the efficacy of DHA as a neuroprotective treatment.
Conclusions
In this study we have characterised a compression model of SCI in the mouse, and shown that it results in a prolonged period of neuronal and oligodendrocyte cell death. In the same mouse model, we have shown that an i.v. bolus of 500 nmol/kg DHA promotes functional recovery and reduces neurofilament loss, neuronal and oligodendrocyte cell death, and microglial/macrophage activation. This study thus provides a detailed validation in a second species (mouse) of the significant neuroprotective
Acknowledgments
We acknowledge the generous support of Chang Gung Memorial Hospital, Taiwan (CMRPG360622). We thank CRODA Healthcare for the donation of DHA oil, and Dr Julian Taylor (Toledo) for assistance with design of the compression platform.
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2018, Prostaglandins Leukotrienes and Essential Fatty AcidsCitation Excerpt :In a mouse model of hypoxic/ischemia (H/I), mice fed a diet with n-3 PUFA supplement (from day 2 of pregnancy to 14 days after parturition) showed amelioration in blood brain barrier (BBB) leakage and decrease in the elevation of matrix metalloproteinase (MMP) activity [194]. In a mouse model of compression spinal cord injury, the transgenic fat-1 mice enriched in omega-3 PUFA showed better outcomes as compared to mice on a high omega-6 diet or a normal diet [195,196]. In another study in which TBI in rats was induced by cortical contusion, intraperitoneal injection of DHA (16 mg/kg) at 5 min after TBI and followed by a daily dose for 3–21 days, was shown to shift microglial morphology from the activated, amoeboid-like state into the surveilling state [197].
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2018, Clinical NutritionCitation Excerpt :Preclinical studies involving rodent compression and hemisection SCI models, at thoracic and cervical levels, have demonstrated that acute DHA injection in the range of 250–500 nmol/kg body weight, with or without sustained dietary supplementation, has a neuroprotective effect and improves neurological outcomes [172–177]. Specifically, DHA administration results in a reduced lesion size and less inflammation (including TNF-α expression), reduced neuronal, oligodendrocyte and neurofilament loss, reduced macrophage/microglia recruitment and activation, and less apoptotic death, and this is correlated with an improved locomotor recovery [172–176]. DHA appears to enhance motor function recovery via its effect on the serotonin fiber input on motor neurons [177].