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Purpose

Limbic epileptogenesis triggers molecular and cellular events that foster the establishment of aberrant neuronal networks that, in turn, contribute to temporal lobe epilepsy (TLE). Here we have examined hippocampal neuronal network activities in the pilocarpine post-status epilepticus model of limbic epileptogenesis and asked whether or not the docosahexaenoic acid (DHA)-derived lipid mediator, neuroprotectin D1 (NPD1), modulates epileptogenesis.

Methods

Status epilepticus (SE) was induced by intraperitoneal administration of pilocarpine in adult male C57BL/6 mice. To evaluate simultaneous hippocampal neuronal networks, local field potentials were recorded from multi-microelectrode arrays (silicon probe) chronically implanted in the dorsal hippocampus. NPD1 (570 μg/kg) or vehicle was administered intraperitoneally daily for five consecutive days 24 hours after termination of SE. Seizures and epileptiform activity were analyzed in freely-moving control and treated mice during epileptogenesis and epileptic periods. Then hippocampal dendritic spines were evaluated using Golgi-staining.

Results

We found brief spontaneous microepileptiform activity with high amplitudes in the CA1 pyramidal and stratum radiatum in epileptogenesis. These aberrant activities were attenuated following systemic NPD1 administration, with concomitant hippocampal dendritic spine protection. Moreover, NPD1 treatment led to a reduction in spontaneous recurrent seizures.

Introduction

Here we examined the neuroprotective bioactivity of NPD1 on limbic neuronal network activities in the pilocarpine model of TLE [] in freely-moving mice using dorsal hippocampal-implanted multi-microelectrode arrays [, ]. We observed that NPD1 reduces brief spontaneous epileptiform field potentials and dendritic spine loss in the dorsal hippocampus, with resultant limited hippocampal epileptic activity. Understanding the cell-type-specific molecular and cellular events underlying NPD1 bioactivity during limbic epileptogenesis will contribute to development of improved therapies for counteracting neuronal network dysfunctions, thus limiting TLE.

In Vivo NPD1 or Vehicle Treatment

NPD1 and vehicle solutions were prepared according to previous protocols [], and then NPD1 (570 μg/kg) or vehicle was administered i.p. once a day for five consecutive days 24 hours after termination of SE.

Rat Mesencephalic TH-Positive Neurons: Primary Culture, Treatment, and Immunocytochemistry

The cultures were performed following the methods described previously (Sun et al. ) with some modifications. Briefly, 1 mm3 of the ventricular mesencephalic region was obtained from Sprague–Dawley rat embryos (embryonic day 15). Two incisions were made, one in the forebrain/mesencephalic limit and the other between the mesencephalon and the hindbrain. Once dissected from the meninges, the tissue was disaggregated using trypsin and DNaseI. Twenty thousand cells were plated in each well of an 8-well chamber slide with B-27/N2 medium containing 2.5 % serum. GDNF, TGF-β, and dcAMP were added once to increase TH-positive cells (Supplementary Fig. 1) and the media were changed every 2 days. The percentage of TH-positive cells achieved was 17–20 %. NPD1 (100 nM) was added to the culture at 7 DIV, 5 min before the addition of 100 nM rotenone, 100 µM MPP+ or 100 µM MPTP. To decide the time of exposure to MPTP and rotenone, 24- and 48-h time points were used (Supplementary Figs. 2, 3). Immunostaining was performed following a previously described protocol (Calandria et al. ). Briefly, cells were fixed with 4 % paraformaldehyde, permeabilized with 1 % Triton and 1 % normal serum in phosphate buffered saline (PBS) for 5 min, and then blocked with 10 % normal serum solution in PBS. The slides were incubated with the primary antibody overnight and then for 1 h with Alexa fluor-conjugated secondary antibodies.

TH-Positive Neuron Cell Death Triggered by Rotenone and MPP+ is Prevented by NPD1

Rotenone and the MPTP metabolite, MPP+, was proposed to induce toxicity primarily by blocking mitochondrial Complex I, which results in the production of superoxide radicals and consequently, oxidative stress (Ramachandiran et al. ). To determine the ability of NPD1 to prevent cell death induced by these three compounds, mesencephalic primary-cultured neurons were exposed to 100 nM rotenone, 100 µM MPTP, or 100 µM MPP+ in the presence and absence of NPD1 for 24 h (Fig. a–c). MPP+ severely decreased the number of TH-positive neurons from 40.3 ± 7.3 in the control to 7.6 ± 1.6; with the addition of NPD1, the number of surviving cells was raised to 14.4 ± 2.7 (Fig. b). MPTP, the precursor of MPP+, also displayed toxicity, promoting the disappearance of 65.7 % of the TH-positive neurons, but this effect was not significantly counteracted by NPD1 (Fig. b). On the other hand, rotenone reduced the TH-positive neuronal population 63 %, and NPD1 reversed that effect almost completely (Fig. c). The NPD1 stereoisomer (10S,17S-diHDHA) had no effect on TH-positive cell survival, indicating that the protection was specifically induced by the S/R stereoisomer (Figs. c, o, p).

Neurons That Survived with the Addition of NPD1 Showed Better Dendritic Morphology on Average

Mitochondrial damage and depletion leads to neurite retraction or shortening (Cherra et al. ). We analyzed the dendritic arbors of surviving TH-positive neurons to evaluate whether NPD1 addition affected neurite integrity. General features were assessed, including maximum dendritic order reached for each TH-positive neuron, number of dendrite segments and branches, the sum of all the segment lengths for each neuron, and the mean for the dendrite diameters. MPTP and MPP+ only altered the parameters related to the length of the dendrites and not related to their thickness, as shown in Fig. d–m. Overall, the integrity of the dendritic arbor was best preserved in cells treated with NPD1 in comparison with the ones that survived without NPD1 treatment (Fig. d–m). Although more TH-positive neurons survived the rotenone treatment with the addition of NPD1 (Fig. c), there was no difference in the maximum dendritic order, the number of segments, or the combined length of all branches and segments when neurons were treated with the lipid mediator (Fig. n, o, q). Intriguingly, the number of branches (Fig. p) on average did not show any significant changes in rotenone-treated TH-positive neurons when compared to controls, suggesting a different mechanism of toxicity. On the contrary, MPP+ induced a reduction in length and number of branches (Fig. d, f, h, j, l), and these effects were reversed almost completely by NPD1. MPTP produced an outcome similar to MPP+, although NPD1 did not provide the same level of improvement (Fig. e, g, i, k, m).

Discussion

NPD1 is a docosahexaenoic acid derivative shown to be protective in neurons and RPE cells undergoing proteotoxic and oxidative stress damage (Mukherjee et al. ; Calandria et al. ) as well as in other dysruptions of homeostasis (Bazan et al. ). Here we report specific NPD1 effects on the survival and dendrite architecture of TH-positive cells exposed to MPTP, its metabolite MPP+, or rotenone. NPD1 increased the survival of TH-positive cells treated with MPP+ (Fig. b) or rotenone (Fig. c). MPTP treatment did not reduce the neuron population to the same extent, and NPD1-mediated neuroprotection was not as strong as with the other two toxins (Fig. b). Interestingly, MPTP toxicity was not reversed by NPD1 to the same extent as MPP+, suggesting that both actions are executed by different mechanisms. MPTP conversion to MPP+ and release occurs in glial cells (Ransom et al. ). It may be possible that this processing of MPTP delays and decreases the amount of MPP+ that reaches neurons, thus causing a weaker but sustained damage compared to the acute insult produced by MPP+. This may explain the differences in TH-positive neurons survival and the protection exerted by NPD1 (Fig. b).

Abstract

Omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) are necessary for functional cell integrity. Preconditioning (PC), as we define it, is an acquired protection or resilience by a cell, tissue, or organ to a lethal stimulus enabled by a previous sublethal stressor or stimulus. In this study, we provide evidence that the omega-3 fatty acid docosahexaenoic acid (DHA) and its derivatives, the docosanoids 17-hydroxy docosahexaenoic acid (17-HDHA) and neuroprotectin D1 (NPD1), facilitate cell survival in both in vitro and in vivo models of retinal PC. We also demonstrate that PC requires the enzyme 15-lipoxygenase-1 (15-LOX-1), which synthesizes 17-HDHA and NPD1, and that this is specific to docosanoid signaling despite the concomitant release of the omega-6 arachidonic acid and eicosanoid synthesis. These findings advocate that DHA and docosanoids are protective enablers of PC in photoreceptor and retinal pigment epithelial cells.

Introduction

In this study, we demonstrate that the free pool size of the essential omega-3 fatty acid DHA is increased to form docosanoids, which play a critical role in survival signaling mechanisms during the protective actions of both in vitro and in vivo retinal PC. We show that, in human retinal pigment epithelial (RPE) cells and PRCs, this resilience is mitigated through 15-LOX-1. We also demonstrate that the neurotrophin pigment epithelium-derived factor (PEDF), which also stimulates docosanoid production, is involved. Moreover, we show that NPD1 prevents the loss of protection bestowed by 15-LOX-1 inhibition in vitro and protects PRCs from light damage in vivo, further signifying that DHA and its docosanoid metabolites are pivotal during RPE and photoreceptor PC.

Abstract

Docosahexaenoic acid, enriched in the brain and retina, generates docosanoids in response to disruptions of cellular homeostasis. Docosanoids include neuroprotectin D1 (NPD1), which is decreased in the CA1 hippocampal area of patients with early-stage Alzheimer's disease (AD). We summarize here how NPD1 elicits neuroprotection by up-regulating c-REL, a nuclear factor (NF)-κB subtype that, in turn, enhances expression of BIRC3 (baculoviral inhibitor of apoptosis repeat-containing protein 3) in the retina and in experimental stroke, leading to neuroprotection. Elucidating the mechanisms of action of docosanoids will contribute to managing diseases, including stroke, AD, age-related macular degeneration, traumatic brain injury, Parkinson's disease, and other neurodegenerations.

DHA arrival to the brain and docosanoids

Pre-clinical events in AD include neuroinflammation, dendritic spine damage, and synaptic dysfunction exacerbated by UOS, which leads to dementia. These events coincide with the decreased DHA content in the brain. A study of brains from early-stage AD patients (with neuronal loss) shows that the amount of DHA and its enzymatic derivative, neuroprotectin D1 (NPD1; 10R,17S-dihydroxy-docosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid), is decreased in the CA1 region of the hippocampus (). NPD1 and other DHA-derived lipid mediators, such as resolvins D1–D5 and maresins, are known as docosanoids and foster overall inflammation resolution (, ).

Significance of bioactive lipids in ischemic stroke

Activation of N-methyl d-aspartate (NMDA) glutamate receptors occurs after stroke and in epilepsy and other neurological diseases (). This activation is accompanied by an increase in membrane phospholipid hydrolysis with concomitant accumulation of free fatty acids (mainly DHA and arachidonic acid) and stearoyl-arachidonoyl-sn-glycerol () due to the rapid activation of phospholipases A2 and C. DHA administration (i.v.) improves behavioral deficit and reduces infarct volume and edema after experimental focal cerebral ischemia (, ). Enzymatic lipoxygenation of DHA in experimental ischemic stroke results in NPD1 formation, which then counteracts pro-inflammatory bioactivity and contributes to sustained neuroprotection (, ). The onset of ischemia causes damage to neurons, and one could argue that in rodent experimental models even after 8 h of reperfusion, accumulation of endogenous neuroprotective mediators (including NPD1) is not sufficient to cope with the resulting damage. Between 24 and 72 h after reperfusion, polymorphonuclear leukocytes migrate to the brain parenchyma, initially removing debris. However, because of their large numbers, these leukocytes induce inflammation and further promote injury, thus increasing stroke size. Continuous injection of NPD1 into the fourth ventricle (400 ng/48 h) after ischemic experimental stroke reduced stroke size in rats by 50% (, ), attenuated polymorphonuclear leukocyte infiltration, and inhibited ischemia induction of cyclooxygenase-2 (COX-2) and nuclear factor κB (NF-κB) (, ). The cerebral ischemic core is surrounded by an area called the penumbra, and due to decreased blood circulation, cell viability is sustained only for 2–4 h post-infarction; therefore, it is critical to protect the penumbra. There is a high correlation between reducing the size of the penumbra and neurological recovery ().