Brain Advance Access originally published online on September 27, 2007
Brain 2007 130(11):3004-3019; doi:10.1093/brain/awm223
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A combination of intravenous and dietary docosahexaenoic acid significantly improves outcome after spinal cord injury
Neuroscience Centre, Institute of Cell & Molecular Science, Queen Mary University of London, UK
Correspondence to: Dr. W. L. Huang, Neuroscience Centre, Institute of Cell and Molecular Science, Queen Mary University of London, 4 Newark Street, E1 2AT, London, UK E-mail: w.huang{at}qmul.ac.uk
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Summary |
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Previous studies have shown that omega-3 polyunsaturated fatty acids such as
-linolenic acid and docosahexaenoic acid (DHA) are neuroprotective in models of spinal cord injury (SCI) in rodents. However, the mechanism of action underlying these effects has not been elucidated, and the optimum treatment regime remains to be defined. We have therefore carried out a detailed analysis of the effects of DHA in adult rats subject to thoracic compression SCI. Saline or DHA (250 nmol/kg) was administered intravenously (i.v.) 30 min after compression. After injury, the saline group received a standard control diet for 1 or 6 weeks, whereas DHA-injected animals received either a control or a DHA-enriched diet (400 mg/kg/day) for 1 or 6 weeks. Other groups received a DHA-enriched diet only for 1 week following injury, or received acute DHA (250 nmol/kg; i.v.) treatment delayed up to 3 h after injury. We also assessed oxidative stress and the inflammatory reaction at the injury site, neuronal and oligodendrocyte survival and axonal damage and the locomotor recovery. At 24 h, lipid peroxidation, protein oxidation, RNA/DNA oxidation and the induction of cyclooxygenase-2 were all significantly reduced by i.v. DHA administration. At 1 week and 6 weeks, macrophage recruitment was reduced and neuronal and oligodendrocyte survival was substantially increased. Axonal injury was reduced at 6 weeks. Locomotor recovery was improved from day 4, and sustained up to 6 weeks. Rats treated with a DHA-enriched diet in addition to the acute DHA injection were not significantly different from the acute DHA-treated animals at 1 week, but at 6 weeks showed additional improvements in both functional and histological outcomes. DHA treatment was ineffective if the acute injection was delayed until 3 h post-injury, or if the DHA was administered for 1 week solely by diet. Our results in a clinically relevant model of SCI show that significant neuroprotection can be obtained by combining an initial acute i.v. injection of DHA with a sustained dietary supplementation. Given that the safety and tolerability of preparations enriched in omega-3 fatty acids is already well-documented, such a combined DHA treatment regime deserves consideration as a very promising approach to SCI management.
Key Words: docosahexaenoic acid; omega-3 fatty acids; spinal cord injury; inflammation; oxidative stress
Abbreviations: AA, arachidonic acid; APC, adenomatous polyposis coli tumour suppressor protein; ß-APP, ß-amyloid precursor protein; BBB, Basso, Beattie and Bresnahan; COX, cyclooxygenase; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; i.v., intravenous; NeuN, neuronal nuclei; 8-OHG, 8-hydroxyguanosine; PBS, phosphate buffered saline; PUFA, polyunsaturated fatty acid; RT, room temperature; SCI, spinal cord injury
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Received June 12, 2007. Revised August 20, 2007. Accepted August 22, 2007.
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Introduction |
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Long-chain omega-3 polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA), are components of membrane phospholipids, and have a structural role in all tissues, including the CNS, but there is also evidence that they have roles in signaling (Salem et al., 2001
). There is also increasing evidence that long-chain omega-3 fatty acids such as DHA and eicosapentaenoic acid (EPA) have a therapeutic potential in various models of neuronal injury (Lauritzen et al., 2000; Blondeau et al., 2002). Spinal cord injury (SCI) affects several million people worldwide, and more than 100 000 new cases are reported each year. Although many promising avenues are being investigated (Thuret et al., 2006), no satisfactory treatment is available at present. Traumatic injury of the spinal cord leads to death of neuronal and glial cells and to degeneration of the ascending and descending tracts. The resulting damage is a consequence of both the primary injury and of the protracted changes described as ‘the secondary injury’ (Tator, 1995). The pathogenesis of secondary injury includes excitotoxicity, inflammation and increased oxidative stress. These events amplify the effect of the primary injury and continue in the days and weeks following the initial insult (Crowe et al., 1997). It is therefore these secondary injury mechanisms, which are the focus of attention for the development of neuroprotective strategies (Baptiste and Fehlings, 2006). There is a need for effective treatments, which address these pathogenetic mechanisms, and are safe and well-tolerated by SCI patients. Recent studies have reported that omega-3 PUFAs are neuroprotective when administered after SCI in rats (Lang-Lazdunski et al., 2003; King et al., 2006). Furthermore, the work led by Bazan and collaborators elegantly demonstrate that metabolites of omega-3 fatty acids have potent neuroprotective properties (Bazan, 2003, 2005, 2006). The safety and tolerability of omega-3 PUFAs is well-documented (Bays, 2006; Mayer et al., 2006) and makes them very strong candidates for rapid translation to the clinic. However, it is essential to characterize the mechanisms that underlie the neuroprotective effects of these compounds, and to define an optimum regime of administration in SCI. We have therefore carried out a detailed analysis of the effects of treatment with DHA in a rat compression SCI model, examining both intravenous (i.v.) and dietary administration, and acute and chronic events. |
Material and methods |
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All experimental protocols were approved by the Animal Care Committee of Queen Mary, University of London, in accordance with the UK Animals Act 1986 and with international guidelines on animal use.
Compression spinal cord injury
Female Sprague-Dawley rats (230–255 g) were deeply anesthetized with 2–3% halothane (Meril, UK) in a mixture of oxygen and nitrous oxide. The spinal cord was injured at thoracic level 12 (T12), using an established static compression model (Nyström et al., 1988; Huang et al., 2007). The skin and muscle overlying the spinal column were incised and a laminectomy was performed at T12, leaving the dura intact. The T11 and T13 spinal processes were clamped in a spinal compression frame, and the compression was applied by suspending the base of the compression platform (area 2 mm x 5 mm) onto the exposed T12 cord under microscopic control. A weight of 50 g was applied statically to the platform for exactly 5 min. Muscle layers were then sutured and skin layers closed with wound clips. Naïve control animals (n = 4) and animals with laminectomy surgery only (n = 4) were also included for histological comparison with injured and treated animals. After surgery, animals were given buprenorphine intramuscularly (0.12 mg/kg; Reckitt Benckiser, UK) and then were placed in warmed cages to recover from anesthesia. Manual bladder expression was performed twice a day for the first week and once daily thereafter, until the establishment of reflex voiding.
Treatment with DHA
SCI surgery was performed in several groups of animals, which received various treatments after injury. In the first and main part of our study, animals received the following treatments: (i) an i.v. injection of saline 30 min after injury and a control diet; or (ii) an i.v. injection of DHA (250 nmol/kg; Sigma, UK) 30 min after injury and a control diet; or (iii) an i.v. injection of DHA (250 nmol/kg) 30 min after injury and a DHA-enriched diet. All i.v. injections were carried out via a tail vein. Animals were sacrificed at 1 week (n = 5 for treatments (i), (ii) and (iii)) or 6 weeks after injury (n = 6 for treatments (i), (ii) and (iii)).
In the second part of our study, we investigated the effect of delaying the acute administration of DHA. Saline (n = 5) or DHA 250 nmol/kg i.v. (n = 5) was injected 3 h after injury. All animals were maintained for 1 week until sacrifice on extended ground maintenance rat chow. We also investigated the effect of omitting acute DHA and administering DHA solely by a DHA-enriched diet for 1 week (n = 6).
Finally, for studies on the mechanism of DHA action, animals received saline (n = 5 for each time point) or DHA 250 nmol/kg i.v. (n = 5 for each time point) 30 min after injury and were sacrificed 1, 3 and 24 h, 3 days or 1 week after SCI. Animals were maintained until sacrifice on extended ground maintenance rat chow.
The control diet consisted of extended ground maintenance rat chow supplemented with sunflower oil (Table 1). Sunflower oil was selected as the base for the control diet, since it does not contain omega-3 fatty acids. The DHA-enriched diet consisted of extended ground maintenance rat chow, to which oil enriched in DHA (Incromega DHA700E SR; Croda Healthcare, England) was added. The oil contained 70–75% DHA and 5% EPA. The individual's daily consumption of chow was monitored prior to the beginning of the study, and the volume of DHA preparation added to the chow calculated correspondingly, so that it resulted in a daily intake of 400 mg DHA per kilogram of animal body mass. Animals consumed slightly less food (
10–15% versus pre-injury state) for 24–48 h after injury, but later they resumed normal consumption of the whole amount of chow provided, and no significant individual differences were seen between animals. An isocaloric volume of sunflower oil was added to the control diet.
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Tissue processing
Animals were overdosed with sodium pentobarbital (60 mg/kg, intraperitoneal; Sagatal, France) and then transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). A 5 mm cord segment containing the compression epicentre was dissected out, post-fixed for 2 h, and then cryoprotected in 30% sucrose in 0.01 M phosphate buffered saline (PBS) overnight at 4°C. Since the laminectomy control animals did not have the compression injury, the 5 mm segment corresponding to the ‘compression area’ was chosen at the level of the laminectomy and processed in a manner identical to the segments dissected in the compression groups. All spinal cord segments were then embedded in OCT medium and stored at –20°C until further processing.
Immunocytochemistry
The spinal cord tissue was cut into 10 µm serial transverse cryostat sections. Selected sections from the compression epicentre were processed for immunocytochemistry. Neuronal Nuclei antigen (NeuN; Chemicon, USA), a general neuronal marker, was used to examine the survival of spinal cord neurons. Adenomatous polyposis coli tumour suppressor protein (APC; Calbiochem, USA), a general oligodendroglial marker, was used to examine the survival of spinal cord oligodendrocytes. ß-Amyloid precursor protein (ß-APP; Zymed, USA) was used to examine axonal injury. It is anterogradely transported under normal conditions, but abnormally accumulates in axons following injury (Li et al., 1995). A clone of mouse anti-rat CD68 (ED1; Serotec, USA) was used to examine macrophage/microglial recruitment. Finally, Hoechst 33342 (Sigma, UK), a fluorescent nuclear dye was used as a counterstain to facilitate the recognition of NeuN, APC or ED1 labelled cells.
The general procedure for immunocytochemistry was as follows. Sections were incubated in 10% normal goat serum for 1 h at room temperature (RT) followed by an overnight incubation with mouse NeuN, APC or ED1 primary antibodies (1:1000, 1:200 and 1:500, respectively) or with rabbit ß-APP primary antibody (1:500) at 4°C. After washes in PBS (10 min 3x), sections were incubated in goat anti-mouse or anti-rabbit secondary antibodies conjugated to tetramethyl rhodamine isothiocyanate (1:400; Jackson Immunoresearch Laboratories, USA) for 2 h at RT. Sections were then washed in PBS before counterstaining with Hoechst 33342 (0.2 mg/100 ml PBS) for 2 min. All primary and secondary antibodies were diluted in PBS containing 0.2% Triton X-100 and 0.1% sodium azide. Slides were mounted in PBS glycerol (1:3) containing 2.5% 1,4-diazobicyclo-(2,2,2)-octane.
Sections were viewed on a Leica epifluorescence microscope (Wetzlar, Germany) using filter blocks for rhodamine or 4',6-diamidino-2-phenylindole. Images were taken using a Hamamatsu C4742-95 digital camera (Herrsching, Germany) and the Leica QWin program (Leica, Germany). Figures were prepared using Adobe Photoshop 7.0.
To determine the numbers of surviving spinal cord neurons and oligodendrocytes, six sections from the epicentre were photographed at 40x magnification using the rhodamine filter block for NeuN or APC. Areas chosen for NeuN were the dorsal and ventral horns (right and left). Areas chosen for APC were the corticospinal tract, the lateral and ventral white matter (right and left). All cells were also visualized using the filter for 4',6-diamidino-2-phenylindole, which showed Hoechst labelled nuclei. In Photoshop, a counting grid was imposed on each image. The number of NeuN or APC immunoreactive cells in each area examined was counted, averaged and expressed as a percentage of that of the naïve animals.
Using the Leica QWin program, ED1 images were obtained at 40x magnification for areas of the dorsal and ventral horns (left and right), the intermediate grey matter (right and left), the corticospinal tract and the lateral and ventral white matter (right and left). Using the same software, the camera image was converted into a binary image of the ED1 labelling. Three measuring frames of identical size (80 µm x 80 µm) were then randomly applied onto each image, and the percentage of the measuring frames covered by this binary image was determined. This was done because the ED1 labelling was too extensive to allow for unambiguous identification of individual cells. The mean of these three measures was then determined for each area per animal.
At 40x magnification, quantification of ß-APP was determined by counting the number of ß-APP immunoreactive axons in three areas of the white matter, namely the dorsal columns, the lateral and ventral white matter (right and left). Regions were analysed from at least three sections per animal.
Behavioural analysis
We used the BBB (Basso, Beattie and Bresnahan; Basso et al., 1996) score to assess the hindlimb locomotor function, including the degree of joint movement, the limb coordination, the plantar placement of paw, the rotation of paw placement, the tail position and trunk stability. The behavioural analysis was carried out double-blind, daily for the first 2 weeks and three times a week thereafter until sacrifice.
Measurement of RNA and DNA oxidation
The nucleic acid (RNA and DNA) oxidation following the compression injury was examined using 8-hydroxyguanosine (8-OHG) immunoreactivity (King et al., 2006). Sections were chosen from the compression epicentre, from animals sacrificed 3 or 7 days after SCI, after having received i.v. saline or DHA (250 nmol/kg) 30 min following SCI and maintained on a normal rat chow diet. The tissue was labelled with mouse 8-OHG antibody (1:1000; QED Bioscience, USA) using the immunocytochemical procedure described above, and then counterstained with the Hoechst dye.
Measurement of lipid peroxidation and protein oxidation
In another set of experiments, we examined lipid peroxidation and protein oxidation following compression SCI. Animals were given i.v. injections of either saline or DHA (250 nmol/kg) 30 min after the compression injury and then were sacrificed at 1, 3, or 24 h after the injections, by inhalation of a high concentration of CO2. Naïve (n = 5) and laminectomy (n = 5 for each time point) animals were also included. A 5 mm cord segment corresponding to the injury epicentre was rapidly dissected out and frozen on dry ice, then stored at –20°C.
The pieces of frozen cord were homogenized in radio immunoprecipitation analysis lysis buffer containing 1% Igepal CA-630, 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulphate in PBS. The buffer also contained phenylmethylsulfonyl fluoride (10 µg/ml), aprotinin (0.1 µg/ml), 100 mM sodium orthovanadate (10 µl/ml) and protease cocktail (5 µl/ml; Sigma, UK). 0.01% w/v 2,6-di-tert-butyl-p-cresol (butylated hydroxytoluene) was added as an antioxidant immediately before homogenization. The protein content of the homogenate was determined by the Bradford assay (Bradford, 1976).
The thiobarbituric acid reacting substance test was used to examine lipid peroxidation, by measuring total thiobarbituric acid reacting substance content in the tissue. The above described homogenate (350 µl) was mixed with 2.1 ml of 1% phosphoric acid (v/v) and the remainder was frozen for subsequent western blot analysis. 2 ml from each sample was then mixed with 0.5 ml of thiobarbituric acid and heated at 95°C for 60 min, cooled on ice, and the adducts were extracted in butan-1-ol. The thiobarbituric acid reacting substance content was measured at 532 nm and the concentration calculated against a standard curve prepared using tetramethoxypropane and thiobarbituric acid (4:1 v/v) and heating at 95°C for 60 min.
For the detection of protein oxidation, an Oxyblot detection kit (Q-biogene, USA) was used to measure the protein carbonyl content in the tissue. A volume of homogenate (described above) corresponding to 10 µg protein was first denatured in 6% sodium dodecyl sulphate and then derivatized by reacting with 2,4-dinitrophenylhydrazine for 15–20 min at RT. Negative controls were reacted with the derivatization-control solution. The reaction was stopped by adding neutralization solution and ß-mercaptoethanol (0.74 M). The samples were then separated on an 8% sodium dodecyl sulphate polyacrylamide gel, and transferred to polyvinylidenedifluoride membranes (Milllipore, USA). The membranes were blocked in Tris-buffered saline (20 mM Tris, 500 mM NaCl, 0.1% Tween 20, pH 7.5; 15 min 3x) containing 5% non-fat milk, and were probed with rabbit anti-2,4-dinitrophenylhydrazine primary antibody (1:150) for 1 h at RT. The membranes were washed as before, incubated in anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:2000; Amersham Biosciences, USA) for 1 h at RT, and then washed again. The bands were visualized by chemiluminescence using an ECL kit and Hyperfilm (Amersham Biosciences, USA). Even protein loading was determined by normalizing the protein content to Coomasie stained protein gels. Samples were quantified by scanning the films and measuring 2,4-dinitrophenylhydrazine content relative to the standard proteins derivatized with 2,4-dinitrophenylhydrazine, using Image J software (NIH, USA).
Measurement of Cyclooxygenase-2
The analysis used the same tissue homogenate as above. Proteins were separated using sodium dodecyl sulphate–polyacrylamide gel electrophoresis on a 10% gel and transferred to polyvinylidenefluoride membranes (Millipore, USA). The membranes were blocked as above and probed for 12 h at 4°C with antibodies against cyclooxygenase-2 (COX-2, 1:1000; Cayman Chemicals, USA). The membranes were then washed in Tris–buffered saline (15 min 3x) and incubated in anti-mouse secondary antibody conjugated to horseradish peroxidase (1:2000; Amersham Biosciences, USA). Following several washes, the bands were visualized as described above. The membranes were then stripped and re-probed with an antibody against ß-actin (1:500; Chemicon, USA) for 12 h at 4°C. The bands were scanned and quantified using Image J software. The results were normalized by subtracting adjacent background levels and expressed relative to the corresponding ß-actin levels.
Statistical analysis
When appropriate, the data were presented as means and SE of the means. One-way, two-way or mixed analysis of variance was used to compare the experimental groups where appropriate. The Tukey–Kramer multi-comparison adjustment was used as the post-hoc test to calculate the significance levels. P < 0.05 was considered statistically significant.
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Results |
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Effects of the DHA injection alone or combined with a DHA-enriched diet
Neuronal survival
Examination of NeuN labelling at 7 days post-injury showed that animals that received a DHA injection alone 30 min after injury or a DHA injection and also a DHA-enriched diet, had substantially more labelled cells in the dorsal (Fig. 1B–D) and ventral horns (Fig. 1F–H) compared to saline-injected animals. Quantitative analyses confirmed these differences, with both groups receiving DHA treatments resulting in significantly more NeuN labelled cells (from 48% to 58% of spinal cord neurons were protected) than saline controls in the dorsal and ventral horns (P < 0.05; Fig. 1I).
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P < 0.05). Scale bar = 50 µm and error bars represent SEM.This neuroprotective effect was also seen at 6 weeks post-injury, with substantially more NeuN-labelled cells present in the dorsal and ventral horns of the two DHA treatment groups compared to saline controls (
52–70% of spinal cord neurons were protected; Fig. 1J). Interestingly, quantitative analysis showed that while both DHA groups again had significantly more neurons in both the dorsal and ventral horns than saline controls (P < 0.05), in the ventral horn the DHA injection plus DHA diet group had significantly more neurons than the DHA injection only group (P < 0.05; Fig. 1J).
Oligodendrocyte survival
Both DHA treatment regimes increased the number of APC labelled oligodendrocytes compared to saline controls (Fig. 2B–D). At 7 days post-injury, there were significantly more APC-positive cells in the corticospinal tract in animals treated with DHA injection alone (27% of oligodendrocytes were protected) or DHA injection combined with the DHA diet (30% of oligodendrocytes) than in the saline group (P < 0.05; Fig. 2E).
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This protective effect was also seen at 6 weeks: both DHA treatment regimes resulted in significantly increased numbers of APC labelled oligodendrocytes in the lateral and ventral white matter at the compression epicentre, compared to saline controls (
26–46% of oligodendrocytes were protected; P < 0.05; Fig. 2F). Interestingly, in the DHA injection plus DHA diet group there were significantly more APC-labelled oligodendrocytes in the lateral white matter compared to animals that received a DHA injection alone (P < 0.05; Fig. 2F). A similar effect was seen in the corticospinal tract at 6 weeks post-injury, with significantly more APC labelled oligodendrocytes (21% protected) present in animals treated with the DHA injection plus DHA diet than in those that received the DHA injection alone (P < 0.05). The analysis of the corticospinal tract was hampered in saline control animals at 6 weeks post-injury because of the expansion of the lesion cavity into this area in many animals.
Axonal injury
At 6 weeks after injury, due to the expansion of the lesion cavity into the dorsal column areas in some of the control animals injected with saline after injury, we were unable to fully investigate the expression of ß-APP in this area. We therefore examined at 6 weeks post-injury, the expression of this axonal injury marker in the relatively better preserved lateral and ventral white matter. Examination showed that in the lateral white matter more ß-APP positive axons were present in the saline-injected control animals (Fig. 3A) compared to both DHA treatment groups (Fig. 3B and C). Quantitative analysis confirmed this reduction in the number of ß-APP positive axons (Fig. 3G) in both DHA groups compared to saline controls. The DHA injection plus DHA diet group also had significantly fewer ß-APP positive axons compared to saline controls in the ventral white matter (P < 0.05), whereas the DHA injection alone group did not differ significantly from either of these groups (Fig. 3D–G).
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Macrophage and microglial recruitment
At 7 days post-injury, there was much less ED1 immunoreactivity in animals injected with DHA than in those injected with saline (Fig. 4A–C). Quantitative analysis showed that DHA treatment groups had significantly lower levels of ED1 immunoreactivity in the dorsal and the ventral horns, the intermediate grey matter, the corticospinal tract and lateral and the ventral white matter, when compared to the saline-injected group (P < 0.05; Fig. 4G). In the corticospinal tract, this protective effect was further significantly enhanced (P < 0.05) by the DHA-enriched diet (Fig. 4G).
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At 6 weeks post-injury, the group of animals treated with an injection of DHA after injury had significantly lower levels of ED1 immunoreactivity when compared to the saline-injected animals (P < 0.05) in the dorsal and the ventral horns, and intermediate grey matter, but not in the lateral and ventral white matter (Fig. 4H). This resulted in a characteristic labelling pattern of ED1 in animals injected with DHA, in that a relatively high density of ED1 immunoreactive cells were seen in a rim around the grey matter (Fig. 4E and F). The decline in ED1 immunoreactivity was even more striking in animals receiving in addition, a DHA-enriched diet; in these animals there was a marked decrease in the grey matter (up to 12-fold) compared to the level in saline-injected animals. There was also a significant decline in ED1 immunoreactivity in the lateral and ventral white matter (P < 0.05 versus saline controls; Fig. 4E, F and H).
Lesion cavity
In our previous studies, we found that following a static compression injury, multiple cavities in the lesion epicentre were not formed until 4 weeks after the injury (Huang et al., 2007). In the current study, we observed no cavities at 7 days post-injury in any of the experimental groups. However, at 6 weeks, multiple cavities were present in the compression epicentre in the saline-treated injured group (19.0 ± 3.4%; Fig. 4D). In striking contrast with the saline group, we did not observe any cavities in animals treated with the DHA injection alone or combined with the DHA diet (Fig. 4E and F).
Behavioural recovery
During the first week post-injury, the DHA injection only group and the DHA injection plus DHA diet group showed greater locomotor recovery than saline-treated controls (Fig. 5, bottom section), and the improved performance of the animals was already apparent at 48 h after injury. At least some plantar placement was present in most animals in both DHA treatment groups by day 7 post-injury, whilst the saline-injected animals only displayed movement of two or three hindlimb joints. Throughout the first week, there was no difference between the two DHA-treated groups. Both DHA-treated groups continued to perform better from the second to the sixth week post-injury (Fig. 5, upper section) compared to the saline group, and by the second week post-injury the DHA injection plus DHA diet group had begun to outperform the DHA injection group. This trend continued and became more marked over the remaining weeks, until the end of the experiment, with most of the animals treated with the DHA injection and DHA-enriched diet able to weight support and plantar step with consistent toe clearance by the sixth week post injury, whilst the animals injected with DHA and on a control diet showed weight support and plantar stepping, but no or only occasional toe clearance. Quantitative analysis confirmed these differences. Both DHA groups showed higher BBB scores from the fourth day to the sixth week post-injury than saline controls (P < 0.05). From the fourth to the sixth week post-injury, the DHA injection plus DHA diet group had a significantly better locomotor performance than the group receiving only a DHA injection (P < 0.05).
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However, when the i.v. injection of DHA 250 nmol/kg was delayed 3 h after injury, the beneficial effect of the fatty acid was lost, and the BBB scores of animals receiving saline or fatty acids were similar at 7 days after injury (3.2 ± 0.3 in DHA-treated animals versus 2.9 ± 0.9 in saline-injected animals). In order to obtain preliminary information on an intermediate time, we also injected DHA 250 nmol/kg at 1 h after injury and we found that the neuroprotective effect of DHA assessed at 7 days post-injury, was still significant (BBB score of 7.3 ± 0.6, compared to the saline-injected rats score of 3.2 ± 0.4; P < 0.05; n = 3–6 animals per group).
In pilot studies, we also investigated the effect of a lower or a higher dose of DHA, i.e. 50 and 2500 nmol/kg, followed by assessment of the animals up to 14 days after injury. No dose-dependence was seen when DHA was injected 1 h after injury, and no overt toxicity appeared in animals injected with the highest dose of DHA (data not shown).
Effects of DHA-enriched diet alone
We did not find any neuroprotective effect conferred by the DHA diet alone at 1 week following compression injury. Neuronal survival was similar to that seen in the saline group, i.e. 40.9 ± 7.1% versus 43.6 ± 9.6% in the dorsal horn and 20.0 ± 6.8% versus 22.9 ± 6.0% in the ventral horn. In addition, there was no significant difference in oligodendroglial survival between the DHA diet alone and saline groups, i.e. 48.4 ± 9.4% versus 40.6 ± 5.0% in the corticospinal tract. However, ED1 labelling was significantly reduced in the ventral horn when compared to the saline group (4.0 ± 0.8% versus 8.9 ± 1.1%, P < 0.05), but only showed a trend towards reduction in the dorsal horn, the intermediate grey matter and the corticospinal tract, and no difference in areas of the lateral and ventral white matter. Behavioural analysis reflected the histology observed: the BBB scores from day 1 to day 7 after injury showed no difference when compared to the saline group (P > 0.05).
Mechanisms underlying the effects of the acute administration of DHA
Lipid peroxidation
In order to define the possible mechanisms underlying the neuroprotective effect of DHA, we measured lipid peroxidation in naïve animals, animals that received a laminectomy, and animals that received spinal cord compression followed by saline or DHA injection (Fig. 6). One hour after surgery, laminectomy induced a slight increase in the level of lipid peroxidation versus naïve controls; the increase was even more marked and significant at 3 h after surgery, whereas by 24 h it had decreased. A different profile was seen after SCI: the injury led to an increase in lipid peroxidation at 1 and 3 h, which was significant at the later time point (P < 0.05 versus naïve). By 24 h after SCI, the level of lipid peroxidation in animals which had received the injury followed by saline was markedly higher than in naive animals or laminectomy controls. Three and twenty-four hours post-injury DHA treatment significantly decreased lipid peroxidation compared to saline controls (P < 0.05). Although DHA reduced the level of lipid peroxidation compared to that seen in saline injected animals at 24 h, both DHA- and saline-treated animals showed significantly more lipid peroxidation than laminectomy controls at this time point (P < 0.05).
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P < 0.05 versus saline at 1 h; 
P < 0.05 versus DHA at 1 h; #P < 0.05 versus DHA at 1 and 3 h; 
P < 0.05 versus saline at 1 h; 
P < 0.05 versus saline at 24 h; ¥P < 0.05 versus laminectomy at 24 h.Protein oxidation
In addition to lipid peroxidation, we examined the effects of the injection of DHA on protein oxidation (20–100 kDa range) following SCI (Fig. 7). Following laminectomy, protein oxidation was increased at both 3 and 24 h after surgery. This increase was amplified by the injury. The administration of DHA after injury markedly decreased the increase in protein oxidation triggered by spinal cord trauma, and this was seen at both 3 and 24 h after injury. The protection conferred by the fatty acid varied across the molecular weight of proteins analysed. At 24 h, DHA reduced the level of oxidation seen in 20–30 kDa proteins to the level seen in controls (Fig. 7D).
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RNA/DNA oxidation
Consistent with our previous findings after hemisection SCI (King et al., 2006
), we observed here a pattern of punctate cytoplasmic labelling of 8-OHG, with no labelling within the nucleus itself (Fig. 8). Most labelled cells were in the grey matter, in particular the ventral horn, while few labelled cells were seen in the white matter. This combined with the morphological appearance of labelled cells suggests that most 8-OHG immunoreactive cells were neurons. This labelling was most prominent at 3 days post-injury (Fig. 8A and B), with both saline- and DHA-injected groups showing a significant drop in the number of 8-OHG immunoreactive neurons in the ventral horn by 7 days post-injury (Fig. 8E; P < 0.05). Between-group comparisons at 3 days post-injury showed that the DHA injection significantly decreased (by 50%) the number of 8-OHG immunoreactive cells in the ventral horn compared to saline controls (P < 0.05; Fig. 8A–C). By 7 days post-injury (Fig. 8C–F), an even greater reduction in the number of 8-OHG immunoreactive cells in the ventral horn was seen in the DHA injection group (by 75%) and in the DHA injection plus DHA diet group (by 80%) when compared to the saline group.
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COX-2 expression
There was a very low basal level of COX-2 expression in the naive spinal cord (Fig. 9). After laminectomy, COX-2 levels increased significantly from 1 to 3 h (P < 0.05), with no further increase at 24 h. In contrast, after SCI followed by saline injection, COX-2 levels not only significantly increased from 1 to 3 h, but also from 3 to 24 h (P < 0.05). DHA treatment resulted in significantly lower COX-2 levels compared to injured saline-injected animals at all time points (P < 0.05); however, the levels of COX-2 were significantly higher than those seen in laminectomy controls, at all time points post-injury.
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Discussion |
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TopSummaryIntroductionMaterial and methodsResultsDiscussionConclusionsSupplementary materialReferences |
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Previously we demonstrated that an acute i.v. injection of DHA administered at 30 min post-injury had significant neuroprotective effects in hemisection and compression models of SCI in rats (King et al., 2006
). In the current study, we have further explored the therapeutic potential of DHA in treating spinal cord trauma and have defined a treatment regime, which leads to significant improvement in both neurological outcome and histological markers of injury after compression of the cord. We have shown that DHA treatment has major neuroprotective effects on white matter tracts and that neuroprotection and functional recovery are greater if acute treatment is combined with chronic administration of DHA in the diet. We have also shown that the acute DHA treatment is still effective if delayed until 1 h after injury, a clinically more realistic time of intervention, and that it is a treatment likely to have a large therapeutic window. Finally, our data show that the administration of DHA leads to a significant reduction in oxidative stress after injury and to a modulation of the inflammatory response after trauma.
DHA increases neuronal survival
In our previous study, we showed that acute DHA treatment increases overall neuronal survival after hemisection and compression injury (King et al., 2006). We have now expanded the analysis in the compression model by showing that i.v. DHA improves neuronal survival in both the dorsal and ventral horns, and that dietary supplementation confers additional protection to ventral horn neurons at 6 weeks. SCI leads to acute and chronic (secondary) neuronal death, which in this compression paradigm, continues for weeks (Huang et al., 2007). One possible mechanism underlying the increased neuronal survival that we observe with DHA is a reduction in glutamate-induced toxicity (Liu et al., 1999). Following traumatic SCI, a massive release of glutamate occurs in the extracellular space in the grey and white matter. Glutamate damages cells by activating N-methyl-D-aspartate receptors and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. This leads to an increased intracellular influx of Ca2+ and activation of phospholipases, proteases and endonucleases, whose activity leads to cell damage and death of neurons (Liu et al., 1999). It has been shown that DHA can reverse glutamate-induced excitotoxicity both in vitro (Wang et al., 2003) and in vivo (Hogyes et al., 2003). DHA may block depolarization-induced increased glutamate efflux and N-methyl-D-aspartate receptor activation partly via inhibition of voltage-sensitive Na+ and Ca2+ channels (Vreugdenhil et al., 1996) and, possibly also via activation of the two-pore domain background K+ channels, i.e. the TREK-1, TREK-2 and TRAAK channels (Lauritzen et al., 2000; Lang-Lazdunski et al., 2003). Most of these events are acute but there are also chronic changes that may be affected by DHA treatment. Ca2+ entry through -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors is regulated by the presence of the GluR2 subunit, which confers Ca2+ impermeability. The GluR2 subunit is decreased following SCI and shows only a slow time course of recovery (Wu et al., 2005). We have shown elsewhere that chronic dietary supplementation with long-chain omega-3 fatty acids can modulate the expression of this subunit, so this may also contribute to the chronic neuroprotective effects of DHA. Ageing is associated with a decrease in GluR2 in selected CNS regions, and this is totally reversed by the chronic intake of a preparation containing DHA (Dyall et al., 2007).
DHA protects white matter tracts
In addition to increased neuronal survival, DHA treatment was seen to improve two indices of axonal integrity, namely oligodendrocyte survival and ß-APP accumulation. It is generally recognized that white matter damage is more critical in SCI than grey matter damage (Eidelberg et al., 1981), and so this effect of DHA is particularly significant. Following compression SCI, Li and colleagues (1995) reported that ß-APP immunoreactive axons appeared within 4 h of injury and remained present throughout the 9-day time course studied. Our study is the first to show ß-APP accumulation at a time period of 6 weeks following compression SCI, and is in accordance with human studies which show ß-APP accumulations in the spinal cord of patients with chronic survival periods of 2, 3 and 50 years (Ahlgren et al., 1996; Cornish et al., 2000).
The present study showed that all DHA-treated animals had significantly increased numbers of oligodendrocytes at 1 and 6 weeks, and reduced levels of ß-APP immunoreactivity at 6 weeks, compared to saline-treated animals. Moreover, at 6 weeks, the effects of the DHA injection and DHA diet was slightly greater than that of the DHA injection alone, suggesting that dietary DHA supplementation may confer increased protection of the white matter. The exact mechanisms of such axonal protection is yet to be determined, but could involve directly the rescue of oligodendrocytes at a distance from the injury site, for example, by reduced excitotoxic oligodendrocyte death, especially in the immediate aftermath of injury (Xu et al., 2004). It may also reflect the general antioxidant and anti-inflammatory effects of DHA (see below).
DHA reduces cavity formation
We demonstrate a significant effect of DHA on the formation of lesion cavities in treatment regimens with the DHA injection alone or combined with the DHA diet. This observation is consistent with our previous finding using the hemisection model of SCI, where a 40–45% reduction in lesion size was observed at 7 days following a bolus i.v. injection of DHA at 30 min after injury (King et al., 2006). We proposed that the reduction in lesion size could be partly due to the increased neuronal and oligodendroglial survival associated with DHA treatment. A similar mechanism may underlie the lack of cavity formation observed in this study. The reduction in secondary cell death can be expected to prevent the enlargement of the initial lesion and the ensuing cavitation. Since inflammation is known to contribute to cell death and cavitation (Fitch et al., 1999), modulation by DHA of the inflammatory response (see below) is also likely to have been a significant contributor.
DHA reduces oxidative stress
The key events initiating cell damage following SCI include Ca2+ influx into cells, impaired mitochondrial function and subsequent generation of reactive oxygen species, especially superoxide anion and hydrogen peroxide (Hall and Braughler, 1986; Amar and Levy, 1999; Park et al., 2004). Moreover, reactive oxygen species can be produced by infiltrated leukocytes following SCI. Consequently, reactive oxygen species attack critical cellular components including phospholipids, proteins and nucleic acids, causing cell dysfunction or death (Floyd and Carney, 1992).
In the present study, we observed an increase in lipid peroxidation and protein oxidation following the sham operation, indicating that laminectomy itself causes a slight increase in oxidative stress, but this event was transient. In contrast, the compression of the cord led to substantial increase in lipid peroxidation and protein oxidation, which were significant at 3 h and greatest at 24 h after injury. These were reflected in global changes in thiobarbituric acid reacting substance, and also in a range of oxidized proteins (our protein analysis reflected protein weight ranges, and could not identify specific proteins, or the absolute levels of expression of the oxidized proteins). These observations are in agreement with previous studies, which showed, for example, significantly increased lipid peroxidation by 6 h after compression SCI (Bao et al., 2004a; Xu et al., 2005). Similarly, increases in protein oxidation have been reported following clip compression SCI in rats (Bao et al., 2004b), with peak oxidation at 24 h, remaining elevated at 72 h post-injury. Thus, it appears that both lipid peroxidation and protein oxidation are early events following spinal cord trauma. Peroxidation of phospholipid fatty acids can cause structural changes to membranes such as increase in membrane viscosity (Dobretsov et al., 1977) and decrease in membrane fluidity (Borst et al., 2000), leading to loss of membrane integrity and subsequent increases in permeability. Oxidative modifications of proteins can occur following lipid peroxidation, which damages cell membranes allowing free radical attack to certain amino acid residues such as arginine, lysine, proline and threonine, producing carbonylated derivatives. In addition, carbonyl groups can be introduced into proteins by reactions with the products of lipid peroxidation (Uchida et al., 1993; Berlett and Stadtman, 1997; Halliwell and Gutteridge, 1997). Here we observed a significant reduction in both lipid peroxidation and protein oxidation in rats treated with a single i.v. bolus of DHA at 30 min after SCI. Nucleic acid oxidation, assessed at 3 and 7 days, was also significantly decreased, consistent with our previous results in hemisection injury (King et al., 2006).
Since oxidative damage is a key early event, which is known to contribute to neuronal death and functional deficits in SCI (Kaptanoglu et al., 2004; Xu et al., 2005), it is likely that reduction of such damage by DHA is a key contributor to its neuroprotective effects. This may be mediated by the effects of DHA on endogenous antioxidant systems. Although we did not directly examine antioxidant effects of the dietary supplementation with DHA following compression SCI, previous animal studies using either ischemic or neurotoxic models of brain injury support this neuroprotective mechanism (Songur et al., 2004; Zararsiz et al., 2006). DHA increases the activity of glutathione peroxidase in vitro (Wang et al., 2003), and superoxide dismutase in vivo (Sarsilmaz et al., 2003; Songur et al., 2004). Interestingly, it has been shown that 10,17S-docosatriene (neuroprotectin D1), a DHA derivative formed during brain ischemia—reperfusion, can prevent damage induced by oxidative stress (Mukherjee et al., 2004).
DHA modulates the inflammatory response after trauma
Another important finding of this study was the effect of DHA on the inflammatory response following the compression injury. SCI results in infiltration of macrophages and activation of microglia at the injury site (Dusart and Schwab, 1994; Fitch et al., 1999), where these cells can clear debris from degenerating cells and myelin, but also secrete a number of proinflammatory cytokines. Although the beneficial effects of inflammation have been emphasized by some authors (Dusart and Schwab, 1994; Schwartz et al., 1999) and implantation of macrophages can promote axonal regeneration (Rapalino et al., 1998), the general consensus is that functional outcome after SCI is improved by therapies that reduce inflammation (Popovich and Jones, 2003; Kwon et al., 2004).
In the current study, we observed a significant reduction in macrophage and microglial recruitment in DHA-treated rats after injury. Furthermore, an additional significant reduction was seen at 6 weeks in DHA-injected rats given dietary DHA when compared to those rats injected with DHA only. Following traumatic SCI, there is activation of phospholipases, which can release arachidonic acid (AA), an omega-6 fatty acid, from membrane phospholipids. AA is released in significant amounts and gives rise to prostaglandin E2 through the activity of COX enzymes (Murphy et al., 1994). We have previously shown that the i.v. injection of AA after spinal cord hemisection exacerbates the effects of injury (King et al., 2006). The recruitment process of macrophages and microglia is partly mediated through the production of highly proinflammatory eicosanoids such as prostaglandin E2, which subsequently enhance vascular permeability, increase local blood flow, increase infiltration of leukocytes and enhance production of proinflammatory cytokines such as TNF-, IL-1 and IL-6. There is abundant evidence that long-chain omega-3 fatty acids such as DHA can inhibit COX activity and the formation of proinflammatory eicosanoids and cytokines (Calder, 2003; Lonergan et al., 2004). Indeed, we have demonstrated in the current study that DHA injected at 30 min after compression SCI significantly reduced the expression of COX-2, a member of the COX family, strongly suggesting that the antagonism of the deleterious cascade of AA may have contributed to the neuroprotective effects of DHA. Moreover, it is likely that sustained dietary DHA also contributed to the additional reduction in macrophage and microglial recruitment seen at 6 weeks, by inhibiting COX-2 function.
In addition to reduction in AA-derived products, DHA and its metabolites may have direct anti-inflammatory effects. The metabolism of long-chain omega-3 fatty acids such as DHA and EPA leads to active metabolites, such as docosatrienes and resolvins (Serhan et al., 2004), which may exert potent effects on the inflammatory response. For example, in vivo and in vitro studies have shown that neuroprotectin D1, a member of the docosatriene family, reduces the expression of proinflammatory cytokines (Lukiw et al., 2005) and reduces IL-1ß mediated COX-2 expression and NF
B activation (Marcheselli et al., 2003; Mukherjee et al., 2004). The reduction by DHA-derived docosanoids of the oxidative stress-induced damage and of the inflammatory response may be involved in the neuroprotective effects of DHA in our compression SCI model (see reviews by Bazan, 2003, 2005, 2006). In a recent study, Belayev and collaborators (2005) have shown that a DHA–albumin complex confers significant neuroprotection after brain ischemia induced by occlusion of the middle cerebral artery. Their study and our observations in SCI (King et al., 2006 and present study) support the significant therapeutic potential of DHA and its neuroprotective derivatives in paradigms of both brain injury and SCI.
SCI also leads to activation of retinoid signaling, and it has been shown that retinoid X receptors appear in the nuclei of macrophages and reactive microglia after injury (Schrage et al., 2006). DHA is an endogenous ligand for retinoid X receptors (de Urquiza et al., 2000), and can activate these receptors at low micromolar concentrations, which are in the range of the systemic circulating concentration of DHA (Lengqvist et al., 2004). This could lead to modulation of the production of proinflammatory cytokines by macrophages and microglia (Kim et al., 2004). Interestingly, it has been reported that 9-cis-retinoic acid, a retinoid X receptor agonist, suppresses the lipopolysaccaharide-triggered inflammatory responses of microglia and astrocytes (Xu and Drew, 2006).
Dietary supplementation with DHA following acute administration confers increased neuroprotection and improved functional outcome
One of the most striking results of this study is that chronic dietary DHA supplementation following an acute i.v. injection led to a better outcome than the i.v. injection alone. This was clearly demonstrated by a number of end points, including neuronal and oligodendrocyte survival, axonal integrity, macrophage recruitment and functional outcome. Behaviourally, rats treated with the DHA injection and DHA diet performed gradually better on the BBB score than the animals treated with the DHA i.v. bolus only; no difference was seen during the first week, but a slight difference in average BBB scores had emerged by week 2, and it became more marked in week 3, and finally significant in the last 3 weeks of assessment (week 4 to week 6).
Although we have not directly measured the degree of DHA uptake, many previous studies have shown that dietary intake of omega-3 PUFAs affects serum and brain levels of fatty acids (Chernenko et al., 1989; Saito et al., 1998). It is likely that the chronic exposure to DHA dietary enrichment has several consequences. First, the sustained intake of omega-3 fatty acids is likely to lead to structural changes in cell membranes in the spinal cord, as we showed in previous studies in the forebrain (Dyall et al., 2007), when aged animals had been treated with DHA and EPA for 12 weeks, at a dose comparable with the present study. Furthermore, in current studies in other models of injury, we have data which show that DHA at the dose of 300 mg/kg already changes significantly the membrane phospholipid composition after 2 weeks of dietary supplementation (data in preparation). All the studies on chronic omega-3 fatty acids supplementation, including ours so far, suggest that after such intervention the omega-6 fatty acid content (i.e. AA content) of the membrane is likely to decrease, in parallel with an increase in DHA content. Furthermore, as suggested by existing microarray studies, the chronic intake of DHA will affect (through retinoid receptors or other transcription factors) the expression of various genes, including genes involved in signaling, inflammation and cytoskeletal structure (Barcelo-Coblijn et al., 2003; Kitajka et al., 2004), and these changes may have a beneficial role in the period post-injury. Previous animal studies using either ischemic or neurotoxic models of brain injury have shown that chronic dietary treatment with omega-3 fatty acids has powerful antioxidant effects (Songur et al., 2004; Zararsiz et al., 2006).
Consistent with the slow onset (later than 1 week) of the improvements conferred by the combined regime, dietary supplementation with DHA for 1 week, without an initial acute bolus, was ineffective. This lack of efficacy may be explained by the fact that oral ingestion did not occur until the first evening after injury, therefore, rather late after the traumatic event. In addition, the return to normal eating patterns (i.e. frequency and quantity) is gradual in rats that have undergone SCI, in particular compression injury. Therefore, it is likely that insufficient DHA reached the spinal cord tissue during the critical time window, and that a rapid intervention with i.v. DHA is essential. Our data supports the idea that the neuroprotection conferred by DHA is complex, and has a ‘fast’ and a ‘slow’ component, which likely act in a synergistic manner.
In this study, the control diet comprised rat chow supplemented with sunflower oil, and contained proportionally more omega-6 than omega-3 PUFAs. Rats, which ingested this diet, recovered in a manner similar to animals placed on a solid control diet, to which no oil had been added (Huang et al., 2007). In particular, the two groups followed a similar pattern of locomotor recovery up to 6 weeks after injury (i.e. the BBB scores of animals having the sunflower oil control diet versus no oil control diet were: 3.7 ± 1.2 versus 3.7 ± 0.6 at 7 days and 13.2 ± 1.9 versus 14.4 ± 0.6 at 6 weeks), and no differences were seen using histological criteria. However, parallel studies in our laboratory have shown that the sunflower oil-supplemented diet is detrimental in our hemisection model of SCI (supplementary data). The reason for this differential effect between the two SCI models is not totally clear but may well reflect subtle differences in the pathogenetic processes in the two models. Hemisection injury causes haemorrhage in the spinal cord. The control sunflower oil-supplemented diet contains omega-6 fatty acids, and these (particularly AA) may worsen the outcome after CNS haemorrhagic injury (Gaetani et al., 1990). In our previous study, we had shown that a bolus of AA worsens outcome after hemisection SCI (King et al., 2006). This suggests that the impact of even small changes in the omega-3–omega-6 PUFA profile of the diet post-injury may be considerable, depending on the type of SCI.
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TopSummaryIntroductionMaterial and methodsResultsDiscussionConclusionsSupplementary materialReferences |
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Our study, in a clinically relevant model of SCI, shows that significant neuroprotection can be obtained by combining an initial acute i.v. injection of DHA with sustained dietary supplementation with this fatty acid. This regime of administration could easily be translated to the clinic. DHA remains efficacious even when the acute i.v. bolus is delayed one hour after injury, which makes this intervention quite feasible for trauma management teams. In the period following SCI, the administration of fatty acids could be maintained either orally or in lipid emulsions, in an injectable form. Such lipid emulsions enriched in fatty acids are already used for nutritional support in various patients, including surgical and critically ill patients, and there is evidence that omega-3 enrichment confers a clear advantage, whereas the presence of omega-6 fatty acids in these emulsions could be deleterious (Calder, 2003
). The remarkable safety and tolerability of preparations enriched in omega-3 fatty acids is now well-documented and thus addresses one of the major concerns associated with any therapeutic intervention in trauma patients. Therefore, the combined regime consisting of an acute bolus of omega-3 PUFA within the first hour after trauma, plus maintenance chronic administration of these fatty acids during recovery deserves consideration as one of the most promising approaches available for SCI management. |
Supplementary material |
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TopSummaryIntroductionMaterial and methodsResultsDiscussionConclusionsSupplementary materialReferences |
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Supplementary material is available at Brain online.
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Footnotes |
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*These authors contributed equally to this work.
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Acknowledgements |
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We gratefully acknowledge support from the BBSRC, St Bartholomew's and The Royal London Charitable Foundation, Croda Healthcare and Corporate Action Trust.
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References |
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