Docosahexaenoic acid pretreatment confers neuroprotection in a rat model of perinatal cerebral hypoxia-ischemia

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BASIC SCIENCE: OBSTETRICS

Docosahexaenoic acid pretreatment confers neuroprotection in a rat model of perinatal cerebral hypoxia-ischemia Deborah R. Berman, MD; Ellen Mozurkewich, MD, MS; YiQing Liu, MD; John Barks, MD OBJECTIVE: We hypothesized that pretreatment with docosahexaenoic acid

RESULTS: DHA pretreatment improved forepaw placing response to

(DHA), a potentially neuroprotective polyunsaturated fatty acid, would improve function and reduce brain damage in a rat model of perinatal hypoxia-ischemia.

near-normal levels (9.5 ⫾ 0.9 treatment vs 7.1 ⫾ 2.2 controls; normal ⫽ 10; P ⬍ .0001). DHA attenuated hemisphere damage compared with controls (P ⫽ .0155), with particular benefit in the hippocampus with 1 mg/kg (38% protection vs albumin controls).

STUDY DESIGN: Seven-day-old rats were divided into 3 treatment

groups that received intraperitoneal injections of DHA 1, 2.5, or 5 mg/kg as DHA-albumin complex and 3 controls that received 25% albumin, saline, or no injection. Subsequently, rats underwent right carotid ligation followed by 90 minutes of 8% oxygen. Rats underwent sensorimotor testing (vibrissae-stimulated forepaw placing) and morphometric assessment of right-sided tissue loss on postnatal day 14.

CONCLUSION: DHA pretreatment improves functional outcome and re-

duces volume loss after hypoxia-ischemia in neonatal rats. Key words: docosahexaenoic acid, hypoxia-ischemia, neonatal, neuroprotection

Cite this article as: Berman DR, Mozurkewich E, Liu Y, et al. Docosahexaenoic acid pretreatment confers neuroprotection in a rat model of perinatal cerebral hypoxia-ischemia. Am J Obstet Gynecol 2009;200:305.e1-305.e6.

C

erebral palsy (CP), an important cause of childhood and lifelong disability, affects approximately 3 per 1000 children by age 8 years.1 Preventive measures, including electronic fetal monitoring, introduced in the 1970s, have proven ineffective. Although there are From the Division of Maternal–Fetal Medicine, Department of Obstetrics and Gynecology (Drs Berman and Mozurkewich), and the Division of Neonatology, Department of Pediatrics (Drs Liu and Barks), University of Michigan Medical School, Ann Arbor, MI. This research was presented at the 29th Annual Meeting of the Society for Maternal– Fetal Medicine, San Diego, CA, Jan. 26-31, 2009. Received Nov. 21, 2008; revised Dec. 28, 2008; accepted Jan. 20, 2009. Reprints not available from the authors. This work was supported through the Rudi Ansbacher Fund at the University of Michigan, the National Institute of Health/National Center for Research Resources Clinical and Translational Science Award Grant UL1RR024986, and the National Institute of Health/National Institute of Neurological Disorders and Stroke Grant 5RO1 NS045812. 0002-9378/$36.00 © 2009 Published by Mosby, Inc. doi: 10.1016/j.ajog.2009.01.020

multiple possible causes of CP including preterm delivery and infection, intrapartum hypoxia-ischemia (HI) represents 1 etiologic subset that might be amenable to either preventative or rescue therapeutic interventions. Biochemically, hypoxic or ischemic injuries of the developing brain involve multiple pathways, including excitotoxicity, intracellular calcium accumulation, free radical generation, nitric oxide–induced tissue injury, proinflammatory cytokines and microglia/macrophages, bioactive lipid mediators, and apoptosis.2 Several animal models have been developed to simulate perinatal brain injury, using ischemic, excitotoxic, and inflammatory stimuli.3-7 These models allow for evaluation of the mechanisms of injury2 and for translational studies of potential therapeutic interventions.5 Docosahexaenoic acid (DHA), an essential long-chain polyunsaturated fatty acid, is found in eggs, fish, fish oils, and algae. Epidemiologic evidence suggests that maternal diets rich in fish are associated with reduced risk for CP, whereas diets rich in meat are associated with increased risk.8 DHA is an especially promising therapeutic or preventive intervention because it may simultaneously exert beneficial effects on several

of the injury cascades contributing to perinatal brain injury, including free radicals, inflammatory cytokines, bioactive lipid mediators, and apoptosis. Research in animal models of Alzheimer disease, stroke, and spinal cord injury has demonstrated neuroprotective and restorative effects of DHA.9-11 Biologic mechanisms whereby DHA might exert its neuroprotective effects include inhibition of cyclooxygenase-2 thereby blocking synthesis of prostaglandins, leukotrienes, and thromboxanes, as well as inhibition of proinflammatory cytokine secretion.11-13 Neuroprotectin D1 (NPD1), a metabolite of DHA, counteracts leukocyte infiltration, nuclear factor-␬B activation, and proinflammatory gene expression in brain ischemia-reperfusion.14 In the prenatal brain, DHA participates in regulation of gene expression, signal transduction, and free radical scavenging.15 However, the role of DHA in perinatal/neonatal HI brain injury is unknown. We used a well-characterized model of unilateral carotid ligation followed by timed hypoxia exposure, which produces unilateral hemisphere HI during hypoxia exposure, followed by reperfusion on return to room air, in neonatal (postnatal day [P] 7) rats.16 Central ner-

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vous system maturation in rats of this age is similar to that of third-trimester human beings.17 In this model, injury severity depends on hypoxia duration, and there is a threshold hypoxia duration below which injury will not occur. The hemisphere opposite the side of the carotid ligation, which is exposed only to transient hypoxia without ischemia, has no discernible neuropathology and can thus serve as a nonlesioned control hemisphere. A variety of indices of ipsilateral hemisphere HI injury severity (compared with nonlesioned contralateral hemisphere) can be used in this model, including hemisphere weight, hemisphere volume, and regional volumes (eg, striatum, cortex, and hippocampus). Functional evaluation of injury severity and of treatment efficacy (eg, by sensorimotor or cognitive testing) is also possible in this model.5 We used this model to test the potential benefit of administration of DHA before neonatal cerebral HI as a neuroprotective strategy. We used a pretreatment strategy to model the clinical scenario in which a preventative treatment could be administered to women presenting with premature labor or with preterm rupture of the membranes; settings putting the fetus at increased risk for adverse central nervous system outcomes as a result of intrauterine or postnatal hypoxia, ischemia, or both.

M ATERIALS AND M ETHODS Preparation of DHA-albumin complex DHA was delivered as a powder (Cat#: D2534 as cis-4,7,10,13,16,19-DHA; Sigma, St Louis, MO). DHA was complexed to human albumin by incubating 4 mL of human serum albumin 25% (Baxter, Deerfield, IL) with 4 mg of DHA to yield a final concentration of DHA 25 mg/25 ␮L. 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 an305.e2

imals in research. Pups were housed with the dam and litter throughout the duration of the experiments. We performed preliminary studies (on 6 litters) that demonstrated that 90 minutes of HI resulted in moderately severe lesions in P7 Wistar rats. In these preliminary studies, neither lesioned nor nonlesioned pups showed ill effects (ie, mortality, decreased weight gain, or temperature alterations) from intraperitoneal (IP) injection of 25% albumin, the vehicle for DHA in this proposal (1.25 or 5 mg/kg ⫽ 5 ␮L/g pup body weight). We also performed studies to determine whether P7 Wistar rats tolerate DHA treatment. DHA was injected IP in the form of a DHA-albumin complex in a dose of 1, 2.5, or 5 mg/kg to P7 rats. These doses were similar to those used in studies of DHA treatment of transient focal cerebral ischemia in adult rats.18 Three control groups received the same volume of albumin without DHA, an equivalent volume of normal saline (NS), or no injection. Immature rat health and physical development were evaluated by measuring temperature, weight gain, and mortality daily during 1 week. Within each litter of 12 pups, 1 male and 1 female pup were allocated to each of these 6 groups, within a total of 3 litters: 6 rats/DHA dose group (ie, a total of 18 DHA-treated rats) and 6 rats/control group. These preliminary experiments demonstrated pup tolerance of DHA; no deleterious effects on daily weight, temperature, or overall mortality were seen. We then proceeded with our goal experiments using 10 litters. Nine of the litters had equal male-female distribution/litter. One litter arrived with 11 pups (6 male and 5 female), giving a total of 119 pups. All 119 pups underwent both HI and treatment interventions on P7. Pups were divided into 3 treatment groups and 3 control groups (n ⫽ 19-20/ group). The 3 treatment groups received DHA 1, 2.5, or 5 mg/kg as DHA-albumin complex, and the 3 control groups received 25% albumin, NS, or noninjection. To avoid confounding treatment with litter, all treatment and control groups were represented in all litters, with 1 pup/sex/group/litter.

American Journal of Obstetrics & Gynecology MARCH 2009

www.AJOG.org After IP injections (5 ␮L/g body weight, 45-93 ␮L), pups were returned to their dams and allowed to recover. At 2.5 hours after injection, pups were anesthetized with isoflurane (induction at 3.5%, maintenance at 1.5%) and the right (R) common carotid artery doubleligated with surgical silk through a ventral neck incision. After surgery, the pups recovered for 1.5 hours: 30 minutes in a 37°C incubator then 60 minutes with the dam for feeding, totaling 4 hours from DHA injection to HI. A time interval of treatment injection 4 hours pre-HI was selected because of existing human data on timing of transplacental passage of acutely dosed DHA.19 After carotid ligation and recovery, HI was induced by placing the pups in 500-mL glass jars partially submerged in a water bath at 36.5°C. The chambers were perfused with a mixture of 8% oxygen balanced with nitrogen for 90 minutes. This hypoxia duration typically produces injury of moderate severity (30-40% reduction in hemisphere or regional volume),5 thus facilitating not only detection of decreased severity of HI injury but also detection of potentiation of injury. After HI, pups recovered in a 37°C incubator for 15-30 minutes until normal activity was resumed, and were then returned to the dam in the home cage, where they remained until P14. Using a 0.6-mm flexible temperature probe (YSI-TeleThermometer; Yellow Springs Instruments, Yellow Springs, OH), animal rectal temperatures were recorded before injection, after injection, before and after carotid ligation, before hypoxia, immediately after HI, 30 minutes after hypoxia, 1 hour after HI, and daily from P8P14. Pups were weighed before HI on P7, and daily from P8-P14. Temperatures and daily weights were used as 2 measures of morbidity. As neonatal rats are in their phase of rapid growth, daily weights serve as a general index of wellbeing. Sick pups typically do not gain weight at the same rate as their healthy counterparts. Temperatures were measured to evaluate whether any observed effects of treatment could be attributable to alterations in body temperature (eg, hypothermia).

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www.AJOG.org Vibrissae stimulation test Sensorimotor functional evaluation was performed on P14 pups using the lateral vibrissae-stimulated forepaw placing test. The vibrissae test was chosen as a readily quantifiable functional measure for injury to the sensorimotor cortex or striatum.20 Tests were conducted by an observer blinded to the treatment group. Vibrissae (whiskers) were unilaterally stimulated on the edge of a surface. At P14, the typical response contralateral to the nonlesioned hemisphere was immediate extension of the forepaw to contact the stimulating surface in 10 of 10 trials. As with human beings having a unilateral brain injury, HI-lesioned P14 rats typically demonstrate a deficit in forepaw placing contralateral to the lesioned hemisphere. Both complete forepaw contacts and partial (incomplete) extension attempts were recorded. A weighted vibrissae score to incorporate data from both complete and partial responses [partial contacts ⫹ 2 * (complete contacts)] was also calculated. Histopathology P14 rats were anesthetized with chloral hydrate (Sigma) before decapitation. Brains removed were immediately placed on dry ice then frozen at -80°C. At 24 hours later, every fourth frozen section (20 ␮m) was collected from anterior to posterior genu of corpus callosum using a cryostat. Sections were subsequently stained with cresyl violet. Using ImageJ software (US National Institutes of Health, Bethesda, MD; http://rsb. info.nih.gov/ij/), severity of brain injury was evaluated by calculating tissue volume. Volumes were calculated from bilateral regional (cortex, striatum, hippocampus, and other) and hemisphere area measurements in regularly spaced coronal sections by summing cross-sectional areas and multiplying by the distance between regularly spaced coronal sections.21 For area measurements the region designated as “other” was defined as all intact-staining hemispheric tissue not included in neocortex, hippocampus, or striatum, and thus included thalamus, septum, fimbria-fornix, corpus callosum, and major white matter tracts. Damage severity of R-sided HI injury

were derived from left (L) and R hemisphere and regional volumes of intact tissue using the formula: % damage ⫽ 100 * (L ⫺ R)/L. Percent protection was calculated using the formula: [(% damage control ⫺ % damage treatment)/% damage control] * 100.

Statistical analysis Differences in percent damage for hemisphere and each brain region (striatum, cortex, hippocampus, other) were evaluated in a linear mixed model analysis of variance with litter as a random effect, treatment and sex as fixed effects, and the treatment by sex interaction. Treatment was evaluated as either 3 DHA levels (1, 2.5, 5 mg/kg) and 3 control groups (NS, 25% albumin, no injection) or pooled treatment levels (DHA pooled, controls pooled). A similar linear mixed model was used to evaluate forepaw placing successes (out of 10 trials) among treatments. Post hoc comparisons of treatment means were carried out using the Tukey-Kramer adjustment for multiple comparisons. Litter effects were evaluated in the linear mixed model using a likelihood ratio test.22 Pearson correlations were estimated between regional damage severity scores; the correlations between brain volume loss by region were very high, ranging from .73-.98. Serial body weights and rectal temperatures were compared among groups by repeated measures analysis of variance.

R ESULTS Bodyweights and temperature All 119 pups tolerated the HI procedures on P7 well. Two of the pups, both from the same litter, were killed by their mother during the 7 days postprocedure. There were no significant differences between treatment and control groups with respect to pretreatment body weight or subsequent serial body weights. There was a statistically significant difference among litters with respect to initial body weight (P ⬍ .0001). There was not a significant difference in pre-HI body weight between males and females. A greater weight gain was seen for males compared with females across all groups (Table 1) (P ⫽ .0114). There were no differences in rectal tempera-

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TABLE 1

Weight gain from postnatal days 7-14, comparing males with females across treatment and control groups Treatment n Malesa

Females

DHA 1 mg/kg

19 12.9 ⫾ .92 12.42 ⫾ .90

DHA 2.5 mg/kg

20 12.15 ⫾ .90 11.70 ⫾ .90

DHA 5 mg/kg

20 12.49 ⫾ .90 11.17 ⫾ .90

Albumin

19 11.52 ⫾ .90 11.31 ⫾ .92

NS

20 12.14 ⫾ .90 10.04 ⫾ .90

No IP

19 12.17 ⫾ .90 11.50 ⫾ .92

...........................................................................................................

...........................................................................................................

........................................................................................................... ........................................................................................................... ........................................................................................................... ...........................................................................................................

DHA, docosahexaenoic acid; IP, intraperitoneal; NS, normal saline. a

P ⫽ .0114 (Tukey-Kramer), comparing males and females across groups.

...........................................................................................................

Berman. DHA acid pretreatment confers neuroprotection in a rat model of perinatal cerebral hypoxia-ischemia. Am J Obstet Gynecol 2009.

tures among groups before treatment, through each stage of the experiment, or during their stay with the dam.

Vibrissae stimulation forepaw placing test In all, 117 animals underwent the lateral vibrissae-stimulated forepaw placing test on P14, 7 days after the injection and exposure to HI. In all animals, R-sided vibrissae stimulation contralateral to the intact hemisphere consistently elicited correct R forepaw placement (10 paw placements in 10 trials). DHA pretreatment in all doses significantly improved contralesional vibrissae forepaw placing to near-normal levels compared with all control groups (P ⬍ .001) (Figure 1). All pairwise comparisons among each of the 3 treatment groups showed statistically significant differences among DHA at each dose and each of the 3 control groups (P ⬍ .0001). There was no statistically significant difference between any 2 adjacent doses (ie, 1 vs 2.5 mg/kg and 2.5 vs 5 mg/kg). There was a statistically significant improvement in forepaw placement at the 1 mg/kg dose compared with 5 mg/kg (P ⫽ .047). No litter or sex effects on forepaw placing responses were seen.

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FIGURE 1

DHA pretreatment attenuates forepaw placing deficits

www.AJOG.org There were no significant sex effects on brain volume loss.

C OMMENT

Performance on vibrissae-stimulated forepaw placing test in treatment and control groups that underwent right (R) carotid ligation followed by 90 minutes of hypoxia at 8% oxygen on postnatal day (P) 7 is shown. Testing was performed on P14 (10 trials/side). The y-axis ⫽ weighted response score in which complete forepaw placing on stimulus surface received score of 2 and partial response (ie, forepaw motion without placing on stimulus surface) received score of 1. Best possible weighted score is 20. Boxes ⫽ interquartile range; horizontal bars ⫽ medians; whiskers ⫽ SE. They extend to data point no more than ⫻1.5 width of box. Because there is minimal variability in treatment groups, circles ⫽ artificial distinction of outlier and stars ⫽ extreme values. All animals consistently responded to R vibrissae stimulation with appropriate R paw placement. Impairment of left (L) paw placement in response to L vibrissae stimulation was seen in controls. Performance was significantly better in all 3 docosahexaenoic acid (DHA) treatment groups (P ⬍ .001; Tukey-Kramer test). Alb, albumin; NaCl, saline; NoIP, no injection. Berman. DHA acid pretreatment confers neuroprotection in a rat model of perinatal cerebral hypoxia-ischemia. Am J Obstet Gynecol 2009.

Histopathology Ninety minutes of HI resulted in moderately severe lesions with striatal and hippocampal atrophy and cortical thinning or cystic infarction in the control groups (Figure 2). Compared with pooled control groups, there was a neuroprotective main effect of DHA pretreatment (pooling all doses) with reduction in ipsilateral hemisphere volume loss (F(1,104) ⫽ 6.06; P ⫽ .0155; F test) and regional effects limited to the hippocampus and other tissues (ie, not cortex, striatum, or hippocampus). Although no significant differ305.e4

ence was found among the different levels of DHA in terms of brain volume loss, higher doses tended to be associated with more brain volume loss than at the lowest dose of 1 mg/kg. (Table 2). When compared with albumin alone, DHA 1 mg/kg pretreatment conferred 38.2% protection against hemisphere volume loss. Litter effects were seen between litters for total brain volume. This effect was significant at all regions (P ⬍ .004) except the striatum (P ⫽ .147). When controlling for body weight differences, a litter effect on total brain volume was still seen in each region.

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The objective of our study was to examine whether pretreatment with DHA has neuroprotective effects against perinatal HI brain injury. We have shown that in all doses, DHA complexed to human albumin provides effective neuroprotection to the neonatal rat brain, improving functional testing to near-normal levels and reducing brain volume loss, particularly in the hippocampus. The neuroprotective effects of DHA demonstrated in this study are similar to those demonstrated in previously published animal models of adult brain injury.9,18 In a differently constructed model of perinatal brain injury, Glozman et al23 demonstrated that intraamniotic injection of ethyl DHA reduced production of thiobarbituric acid-reactive substance, a marker of oxidative injury, in the fetal brain after in utero ischemic stress. To our knowledge, our study is the first to demonstrate that DHA pretreatment improves functional outcome after perinatal cerebral HI. If studies using a wider battery of neurobehavioral tests confirm this preliminary result, effects should be sustained. In addition, this improvement in function may be of greater translational importance than changes in histopathology. Nevertheless, our study provides evidence that DHA pretreatment also decreases brain volume loss secondary to hypoxic injury. Our finding that lower doses of DHA produced a trend to less brain volume loss and higher vibrissae score than higher DHA doses was unexpected. However, in an adult rat model of stroke, Belayev et al18 also demonstrated a reverse dose dependency, with more favorable results found with the lower of 2 tested DHA doses, 2.5 mg/kg. As in the latter study, the current work does not elucidate the mechanism underlying this reverse dose dependency. One possible explanation could be that at higher doses, DHA might act as a substrate for lipid peroxidation-mediated injury mechanisms.18

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www.AJOG.org In our study, the predominant histopathologic effect of DHA treatment appears to have been in the hippocampus and in the other tissues, which includes the hippocampal connections in the fimbria-fornix. Unlike most brain regions, the hippocampus has intrinsic neuroregenerative potential because it incorporates a region, the dentate gyrus, in which active neurogenesis continues, even into adulthood.24 Evidence that DHA can promote hippocampal neurogenesis and neurite outgrowth25,26 suggests that these effects might explain, at least in part, the apparent region-specific beneficial effect of DHA in HI rats. Of interest is the significant functional improvement in the vibrissae score with DHA despite minimal histopathologic improvement in the striatum and cortex. Further research is necessary to elucidate the mechanism of this observed functional improvement. A limitation of this study is that our model may not fully replicate the perinatal conditions leading to brain injury and subsequent neurodevelopmental disability because the mechanism of injury in this model was HI alone without an added inflammatory stimulus. Likewise, our histopathologic methods did not permit evaluation of hippocampal neurogenesis. Our study was limited to observation of clinical effect in an animal model and did not explore the mechanism of action of the observed benefits. DHA is the biochemical precursor of several biologically active lipid mediators, classed neuroprotectins, neuroprostanes, and resolvins.11,27,28 NPD1, in particular, has been shown to inhibit leukocyte infiltration and activation of microglia in an adult rodent model of brain ischemiareperfusion. NPD1 blocks proinflammatory gene expression, down-regulating the synthesis of proinflammatory cytokines interleukin 1␤ and tumor necrosis factor-␣.11,29 Similarly, in an in vitro model of cultured microglial cells, De Smedt-Peyrusse et al30 demonstrated that DHA pretreatment alters Toll-like receptor 4 location at the microglial cell membrane, thereby down-regulating proinflammatory cytokine synthesis in response to inflammatory stimuli. DHA

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FIGURE 2

DHA pretreatment decreases hypoxic ischemic damage

Comparison of histopathology in 2 postnatal day (P) 14 rat pups that underwent right (R) carotid ligation plus 90 minutes of 8% oxygen exposure on P7 after receiving A and C, 25% albumin pretreatment or B and D, Docosahexaenoic acid (DHA) 1 mg/kg pretreatment. Corresponding coronal sections were cresyl violet stained. In albumin control, ipsilateral cerebral hemisphere damage is evident; there is prominent striatal atrophy (S) with adjacent lateral ventriculomegaly (*), marked hippocampal atrophy (arrow), and cystic infarction of parietal cortex (arrowhead). In comparison, in DHA-treated animal, D, there is subtle hemispheric asymmetry with smaller R hemisphere and less prominent hippocampal atrophy (arrow) compared with control. (Scale bar ⫽ 1 mm). Berman. DHA acid pretreatment confers neuroprotection in a rat model of perinatal cerebral hypoxia-ischemia. Am J Obstet Gynecol 2009.

has also been shown to inhibit excitotoxicity and apoptosis,11,14,15,31,32 suggesting that the observed neuroprotective effect probably results from simultaneous

activity at several stages of the brain injury cascade. Further studies are needed to test the potential benefit of DHA in a more com-

TABLE 2

Effect of docosahexaenoic acid pretreatment on brain damage severity by region n

Hippocampus

Othera

DHA 1 mg/kg

19

42 ⫾ .30

19 ⫾ .16

DHA 2.5 mg/kg

20

DHA 5 mg/kg

20

Albumin

19

67 ⫾ .15

31 ⫾ .11

45 ⫾ .26

41 ⫾ .16

NS

20

59 ⫾ .17

31 ⫾ .14

51 ⫾ .27

43 ⫾ .16

No IP

19

55 ⫾ .21

28 ⫾ .12

40 ⫾ .23

34 ⫾ .19

Treatment

b

b

Cortex

Striatum

30 ⫾ .28

28 ⫾ .20

..............................................................................................................................................................................................................................................

49 ⫾ .37

28 ⫾ .17

48 ⫾ .28

38 ⫾ .19

..............................................................................................................................................................................................................................................

45 ⫾ .31

21 ⫾ .14

37 ⫾ .27

36 ⫾ .20

.............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. .............................................................................................................................................................................................................................................. ..............................................................................................................................................................................................................................................

DHA, docosahexaenoic acid; IP, intraperitoneal; NS, normal saline. Values are means ⫾ SD of regional percent damage, calculated from regional volumes using the formula: 00 * (L ⫺ R)/L. a

“Other” refers to any intact standing tissue other than hippocampus, cortex, or striatum; b P ⬍ .05 compared with pooled controls (Tukey-Kramer).

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Berman. DHA acid pretreatment confers neuroprotection in a rat model of perinatal cerebral hypoxia-ischemia. Am J Obstet Gynecol 2009.

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plex model of perinatal of HI potentiated by intrauterine inflammation. Likewise, studies testing the durability of the neuroprotective effects of DHA over a longer postinsult survival time are needed. Future studies also need to characterize the molecular mechanisms whereby DHA exerts its benefit to the HI developing brain. In summary, this study demonstrates that DHA pretreatment reduces brain volume loss and improves contralateral vibrissae-stimulated forepaw placing test results to near-normal levels in neonatal rats undergoing HI. The most promising aspect of this research is the observed functional improvement. Further animal studies are needed to elucidate the mechanism of this neuroprotection. Experiments to establish the durability of this finding will be necessary before clinical studies of DHA supplementation for neuroprotection in pregnancies at high risk for adverse neurodevelopmental outcome can be undertaken. f ACKNOWLEDGMENTS The authors thank Edward D. Rothman, PhD, and Kathleen B. Welch, MPH, MS, at The University of Michigan Center for Statistical Consultation and Research for statistical advice and assistance, and Joyce Li for assistance with histopathology.

REFERENCES 1. Bhasin TK, Brocksen S, Avchen RN, Van Naarden BK. Prevalence of four developmental disabilities among children aged 8 years—metropolitan Atlanta developmental disabilities surveillance program, 1996 and 2000. MMWR Morb Mortal Wkly Rep 2006;55:1-9. 2. Grow J, Barks JD. Pathogenesis of hypoxicischemic cerebral injury in the term infant: current concepts. Clin Perinatol 2002;29:585-602. 3. Skoff RP, Bessert D, Barks JD, Silverstein FS. Plasticity of neurons and glia following neonatal hypoxic-ischemic brain injury in rats. Neurochem Res 2007;32:331-42. 4. Hagberg Peebles D, Mallard C. Models of white matter injury: comparison of infectious, hypoxic-ischemic, and excitotoxic insults. Ment Retard Dev Disabil Res Rev 2002;8:30-8.

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