Objective
Lipopolysaccharide pretreatment potentiates hypoxic ischemic injury. We hypothesized that docosahexaenoic acid pretreatment would improve function and reduce brain volume loss in this rat model of perinatal brain injury and inflammation.
Study Design
Seven-day-old rats were divided into 3 groups: intraperitoneal docosahexaenoic acid 1 mg/kg and lipopolysaccharide 0.1 mg/kg, 25% albumin and lipopolysaccharide, and normal saline. Injections were given 2.5 hours before carotid ligation, followed by 90 minutes 8% O 2 . Rats underwent sensorimotor function testing and brain volume loss assessment on postnatal day 14.
Results
Docosahexaenoic acid pretreatment improved vibrissae forepaw placing scores compared with albumin/lipopolysaccharide (mean ± standard deviation weighted score/20: 17.72 ± 0.92 docosahexaenoic acid/lipopolysaccharide vs 13.83 ± 0.82 albumin/lipopolysaccharide; P < .007). Albumin/lipopolysaccharide rats scores were worse than those of the normal saline/normal saline rats (13.83 ± 0.82 vs 17.21 ± 0.71; P = .076). No significant differences in brain volume loss were observed among groups.
Conclusion
Lipopolysaccharide inflammatory stimulation in conjunction with hypoxic ischemic resulted in poorer function than hypoxic ischemic alone. Docosahexaenoic acid pretreatment had significantly improved function in neonatal rats exposed to lipopolysaccharide and hypoxic ischemic.
Intrapartum hypoxia-ischemia (HI) may lead to neonatal encephalopathy and to permanent neurologic disability. Electronic fetal monitoring (EFM) was introduced in the 1970s with the expectation that recognition of intrapartum hypoxia and timely intervention would reduce cerebral palsy by as much as 50%. Unfortunately, use of EFM failed to reduce cerebral palsy in large randomized controlled trials, and the widespread use of EFM and expedited delivery in developed countries has not reduced the incidence of cerebral palsy among term infants. One possible reason for the ineffectiveness of EFM in preventing cerebral palsy may be the complex and previously poorly understood cause of neurologic injury.
Recently, the role of inflammation and the fetal systemic inflammatory response syndrome in the cause of cerebral palsy has been recognized. Using a large California cord blood repository, Nelson et al demonstrated that increased levels of interleukins (ILs) 1, 6, 8, 9, 11, 13, and tumor necrosis factor-α (TNF-α), were present in cord blood taken from term infants destined to have cerebral palsy develop compared with healthy controls. Similarly, in a prospective cohort study of 123 preterm infants born to mothers who underwent amniocentesis, Yoon et al demonstrated that elevations of amniotic fluid IL- 6 and -8 as well as histologic funisitis were strongly associated with the diagnosis of cerebral palsy at 3 years of age. A population-based study demonstrated that the coexistence of a potentially asphyxiating condition, such as tight nuchal cord and maternal infection, conferred a much higher risk of spastic quadriplegic cerebral palsy together than either condition alone. Drawing on these observations, Peebles and Wyatt hypothesized that inflammation lowers the threshold at which intrapartum hypoxia results in neurologic injury. This improved understanding of the interaction between hypoxia and inflammation suggests new approaches to perinatal neuroprotection.
One such novel approach is docosahexaenoic acid (DHA), a long chain polyunsaturated fatty acid. DHA is an integral component of neuronal cell membranes and synaptic terminals. DHA is readily available in the diet in fish and algae, and epidemiologic observation suggests that maternal diets rich in fish are associated with reduced risk for cerebral palsy. In adult rodent models of brain ischemia-reperfusion and spinal cord injury, DHA has been shown to exert neuroprotective effects and to improve functional outcome. DHA may exert antiinflammatory effects by altering the presentation of Toll-like receptor 4, the lipopolysaccharide (LPS) receptor, on the microglial cell membrane, thereby modulating the cyclooxygenase-2 signaling pathway, and reducing proinflammatory cytokine production. DHA is the metabolic precursor of D-series resolvins and neuroprotectins. Neuroprotectin D1 attenuates NFκB production and cyclooxygenase-2 expression, reduces influx of polymorphonucleocytes, and counters apoptosis, thus promoting neural cell survival. D-series resolvins block TNF-α induced IL-1β transcripts in microglial cells and limit polymorphonucleocyte infiltration into inflamed brains.
In our earlier experiments using a neonatal day 7 (P7) rat model of perinatal HI, we have shown that DHA pretreatment with 1 mg/kg, 2.5 mg/kg, and 5 mg/kg doses reduces brain volume loss and improves neurologic functioning as measured by the vibrissae-stimulated forepaw placing test. In those dose-finding experiments, we demonstrated that the 1 mg/kg dose was most neuroprotective.
The objective of this second series of experiments is to test DHA pretreatment in a neonatal rat (P7) model of HI potentiated by inflammation. We hypothesized that DHA pretreatment would reduce brain volume loss and improve neurologic functioning in an animal model of perinatal HI with inflammation that may more nearly reflect the conditions leading to cerebral palsy than HI alone.
Materials and Methods
Preparation of DHA-albumin complex
DHA was delivered as a powder (cat. no: D2534 as cis-4,7,10,13,16,19-DHA; Sigma-Aldrich, St. Louis, MO). DHA was complexed to human albumin by incubating 4 mL human serum albumin 25% (Baxter, Deerfield, IL) with 4 mg of DHA to yield a final concentration of DHA 25 mg/25 μL. Each vial was aliquoted in 1-mg/mL samples and kept under nitrogen in a −20°C freezer. Nitrogen was reapplied to the vials weekly.
Animals
P7 Wistar rats were obtained in litters adjusted to equal sex distribution (Charles River Laboratories, Portage, MI). Animals were treated in accordance with protocols approved by our University Committee on the Use and Care of Animals in research. Pups were housed with the dam and littermates throughout the duration of the experiments.
To test the effect of DHA pretreatment, we used a modification of the LPS pretreatment model described by Eklind et al. In contrast to the Eklind et al model, we chose the 90-minute duration of hypoxia for consistency with our prior studies with DHA using HI alone. Based on our prior dose finding studies, we used the 1-mg/kg dose of DHA for pretreatment in the current experiments. Sample size calculations indicate that 16-17 subjects/group are needed to detect a reduction in percent damage of 1 standard deviation (SD), with α = .05, β = 0.8.
On the basis of these calculations, 6 litters (n = 72) of Wistar pups all underwent both HI and treatment interventions on P7. Within each litter, pups were divided into 3 groups. The first group received intraperitoneal (IP) DHA 1 mg/kg as DHA-albumin complex, immediately followed by IP LPS (0.1 mg/kg) (DHA/LPS). The second group received IP 25% albumin, immediately followed by IP LPS (ALB/LPS). The third group received IP normal saline (NS), immediately followed by IP NS (0.1 mg/kg) (NS/NS). This third group (NS/NS) was used as a control to provide lesioning conditions within the experiments from which we could compare histopathologic lesions in a group of pups undergoing HI alone without an inflammatory stimulus (LPS).
Pups received the initial IP injection (5 μL/g body weight) of DHA, 25% albumin, or NS, followed immediately by IP Escherichia coli LPS 055:B5 (0.1 mg/kg) (Sigma-Aldrich) or NS injections. Pups were returned to their dams and allowed to recover. At 2.5 hours after injection, pups were anesthetized with isoflourane (induction at 3.5%, maintenance at 1.5%) and the right common carotid artery was divided after double ligation with surgical silk through a ventral neck incision. Pups then recovered for 1.5 hours (30 minutes in a 37°C incubator, then 60 minutes with the dam). After carotid ligation and recovery, HI was induced by placing the pups in 500-mL glass jars partially submerged in a water bath at 36°C in 8% oxygen for 90 minutes to induce unilateral cerebral HI. After HI, pups recovered in a 37°C incubator for 15-30 minutes. Once normal activity was resumed, the pups were returned to the dam where they remained until P14.
Animal rectal temperatures were obtained using a 0.6-mm flexible temperature probe (YSI-400 Tele-Thermometer; Yellow Springs Instruments, Yellow Springs, OH) before injection, after injection, before and after carotid ligation, before and after HI, 1 hour after HI, and on P14. Pups were weighed before HI on P7 and subsequently on P14. The temperatures and weights were obtained both as representative measures of morbidity and to document that there were no temperature effects of the treatments, that is, hypothermia that could affect or improve outcomes.
Vibrissae-stimulated forepaw placing test
We chose the vibrissae-stimulated forepaw placing test as a readily quantifiable functional measure of injury to the sensorimotor cortex or striatum. The vibrissae test was performed on P14 pups in the following manner: stimulation of the vibrissae (whiskers) on a surface edge results in extension of the forepaw on the same side as the simulated whiskers to reach the stimulating surface. In a partial response, the forepaw is incompletely extended without contacting the stimulating surface. Therefore, stimulation of the left vibrissae can demonstrate left forepaw placement deficits as a consequent of right-sided hypoxic ischemic injury. As the right hemisphere controls left-sided motor function, stimulation of the left vibrissae tests the right hemisphere. Stimulation of the right vibrissae, contralateral to the intact hemisphere, tests the hemisphere without pathology. A weighted vibrissae score to incorporate data from both complete and partial responses was calculated using the formula [partial contacts + 2 × (complete contacts)].
Histopathology
P14 rats were deeply anesthetized and euthanized via decapitation, followed by brain removal. Frozen coronal 20 μm sections were stained with cresyl violet. Severity of brain injury was evaluated by calculating volume of tissue with intact staining using ImageJ software ( http://rsb.info.nih.gov.easyaccess1.lib.cuhk.edu.hk/ij/ ; National Institutes of Health, Bethesda, MD). Volumes were calculated from hemispheric and regional (cortex, striatum, hippocampus, and “other”) area measurements in regularly spaced sections. The “other” region included thalamus, septum, fimbria-fornix, corpus callosum, and major white matter tracts. Damage severity (percent tissue loss) of right-sided injury was calculated as previously described. Injury was also evaluated using a semiquantitative severity score by an observer unaware of animal treatment identity in at least 15 sections/brain, in 7 regions: striatum, cortex at the level of the striatum, thalamus, cortex at the level of hippocampus, and regions CA1, CA3, and dentate gyrus of the dorsal hippocampus. Pathology was rated in each region on a scale of 0-4 in increments of 0.5, with 0 representing no detectable damage and 1-4 representing <10%, 20-30%, 40-60%, and >75% tissue infarction (or selective neuronal necrosis in the hippocampus), respectively, and scores were summed across regions. This scoring approach was modified from Thoresen et al.
Statistical analysis
A linear mixed models analysis of variance was used to evaluate differences in percent damage for hemisphere and each brain region among groups. We used litter as a random effect, treatment and sex as fixed effects, and evaluated treatment by sex interaction. A similar linear mixed model was used to evaluate forepaw placing successes among treatments. Post hoc comparisons of treatment group means were carried out using the Tukey-Kramer adjustment for multiple comparisons. The effect of LPS on mortality was evaluated by analysis of variance with treatment as a fixed effect and litter as a random effect. NS/NS control pups were excluded from subsequent mortality analysis, as there were no deaths in this group. To evaluate an effect of DHA on mortality among all LPS-treated pups, we used a generalized mixed model with treatment as a fixed effect and litter as a random effect to assess the probability of dying in the ALB/LPS and DHA/LPS treatment groups only.
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