In a recent study published in Biomedicine and Pharmacotherapy, researchers described a novel therapeutic approach for neonatal hypoxic-ischemic (HI) brain injury.
Study: Omega-3 fatty acid diglyceride emulsions as a novel injectable acute therapeutic in neonatal hypoxic-ischemic brain injury. Image Credit: Sergii Sobolevskyi/Shutterstock.com
Background
Neonatal HI encephalopathy occurs due to a lack of oxygen and blood flow to the brain during labor/delivery. It is among the major causes of cerebral palsy, death, and other neurological conditions.
Therapeutic hypothermia (TH) is the standard of care for HIE. However, TH has several limitations and challenges in clinical practice, such as cognitive impairments, variable clinical response, and incomplete protection, warranting more effective and accessible therapies for HIE.
Studies have provided evidence for dietary supplements and oral omega-3 fatty acids (n-3 FAs) as potential neuro- and cardioprotective strategies.
Previously, the researchers showed that administering docosahexaenoic acid (DHA) as triglyceride (TG) emulsion particles following HI injury in neonatal mice significantly attenuated brain damage.
About the study
In the present study, researchers evaluated the efficacy of a novel n-3 diglyceride (DG) lipid emulsion in neonatal HI brain injury. They produced n-3 DG oils through reverse glycerolysis reactions and prepared 10% lipid emulsions by mixing n-3 DG or TG-DHA oils with egg yolk phosphatidylcholine.
Next, the average particle size, polydispersity index (PDI), and zeta potential of emulsions were determined. Oxidative measurements were made using the p-anisidine assay.
The emulsions were incubated with a buffer with or without bovine lipoprotein lipase (LpL). Human plasma containing apolipoprotein C-II was added to the mixture. Released free FA (FFA) was measured.
Experiments were performed with increasing LpL levels. C57BL/6J neonatal mice (eight days old) were intraperitoneally injected with DG or TG emulsion; changes in plasma glyceride levels were evaluated. Blood samples were collected after injection.
In addition, another group of mice aged 10 days were subjected to HI insult. Mice received two doses of DG or TG emulsion, one shortly after the HI insult and the other an hour later; controls were injected with saline (two doses).
Further, Wistar rat pups aged seven days also underwent a similar procedure for HI brain injury. Rats received a single dose of saline, n-3 DG, or Omegaven (commercial TG-based emulsion) immediately after the HI insult.
One group of rats received TH for five hours post-HI insult. Negative geotaxis and righting reflex performances were evaluated 24 hours after the injury. Twenty-four hours after reperfusion, mouse brains were obtained (immediately after behavioral tests), and infarct sizes were computed.
Eight days after reperfusion, rat brains were obtained; hematoxylin and eosin staining were performed to analyze brain area loss. Immunofluorescence was performed for astrocyte and microglia markers.
Findings
n-3 DG preparations had smaller particle sizes than Omegaven or n-3 TG. n-3 DG and n-3 TG emulsions were homogeneous with low PDI values. Further, n-3 TG emulsions showed FFAs at 4–8 weeks, suggesting spontaneous hydrolysis, whereas n-3 DG emulsions did not.
In addition, there was no detectable deterioration in the DG emulsion at six months. The p-anisidine values of all oils and emulsions were below 20 mEg/l.
The zeta potential was -50 mV for DGs and -35 mV for TGs. Basal FFA levels (without LpL) were similar between DG and TG emulsions. The highest lipolysis was observed in n-3 DG emulsion, with over 1.5-fold more FFAs released than n-3 TG emulsions.
Plasma glyceride levels in neonatal mice were substantially elevated one hour after n-3 DG injection; glyceride levels peaked at two hours with a three-fold increase compared to baseline and returned to baseline levels by four hours.
In contrast, glyceride levels at one hour after n-3 TG injection were similar to baseline levels but increased at later time points (2h and 4h).
Further, neonatal HI injury mice treated with n-3 DG showed a significant decline in infarct size (87%), whereas n-3 TG treatment reduced the damage by 43%. Likewise, n-3 DG was the most effective in the rat model.
Moreover, the reflex performance of neonatal HI mice after n-3 DG treatment was similar to age-matched naïve mice, suggesting that n-3 DG preserved neurofunctional outcomes.
In the rat model, astrogliosis was significantly reduced seven days after HI injury with n-3 DG treatment compared to saline. Moreover, microgliosis was also significantly attenuated in the n-3 DG treatment group relative to the saline group.
Conclusions
The researchers showed that n-3 FAs in DG lipid emulsions are more beneficial than n-3 TG in reducing brain injury.
The n-3 DG emulsion was superior in decreasing infarcts than TH, the current standard of care, and it also attenuated astrogliosis and microgliosis during the sub-acute phase of the injury. Therefore, n-3 DG confers neuroprotection and activates cytoprotective mechanisms in response to brain injury.