Magnesium sulfate reduces inflammation-associated brain injury in fetal mice fetal anoxia

INTRODUCTION

Cerebral palsy (CP) is a non-progressive motor impairment syndrome which occurs in 1 to 3.6 per 1000 live births. 1 – 3 almost 8% of ex-preterm children born at less than 28 weeks of gestation are affected by CP. 2 , 4 , 5 despite the advances in perinatology and neonatology and the dramatic reduction in the mortality of high-risk infants, there has been no reduction in prevalence of CP. 6

Magnesium sulfate (mgso 4) has been investigated in several clinical trials as well as in systemic reviews and meta-analyses as a possible therapeutic agent to reduce CP in “at-risk“ fetuses. 7 – 11 in most of these trials, “at risk” fetuses were those likely to be born preterm from spontaneous preterm birth—a condition that is frequently associated with intrauterine inflammation.Fetal anoxia


while these studies demonstrated that mgso 4 may prevent CP at 2 years of life, there have been some concerns raised with the use of antenatal mgso 4. Although a meta-analysis did not show an increase in neonatal death associated with mgso 4, 8 there still has been some concern raised regarding a possible increased risk of neonatal death from mgso 4 12 , 13 furthermore, in an in vivo model, maternal administration of high doses of mgso 4 was shown to lead to cell death in a developing mouse brain. 14

Based on the latest cochrane review, 9 the neuroprotective role for antenatal mgso 4 given to women at risk of preterm birth for the preterm fetus is “now established.” despite these recommendations, the mechanism by which mgso 4 serves as a neuroprotective agent in the preterm brain has not been elucidated.Fetal anoxia

From models of hypoxic-ischemic (HI) and traumatic brain injury, the protective effect of mgso 4 is believed to be through its action as a non-competitive antagonist of the N-methyl-D-aspartic acid (NMDA)-receptor. 15 – 19 however, other animal data suggest that mgso 4 may serve an anti-apoptotic role and prevent neuronal cell loss. 20 , 21 to date, there are no animal trials investigating the use of mgso 4 as a neuroprotective agent in the setting of prenatal inflammation.

These studies sought to determine whether mgso 4 administered to the mother can prevent fetal brain injury as a possible mechanism by which it appears to be neuroprotective in human clinical trial of preterm infants. Using a mouse, we have demonstrated that intrauterine inflammation results in a cytokine response in the fetal brain, white matter damage (WMD) as well as neuronal injury. 22 – 25 using this established model, 26 we investigated the ability of mgso 4, administered to the mother, to prevent fetal brain injury.Fetal anoxia the objectives of these studies were to investigate: 1) whether administration of mgso 4 altered the pro-inflammatory response in fetal brain; 2) whether administration of mgso 4 altered apoptotic or necrotic pathways in the fetal brain and, most importantly, 3) whether mgso 4, administered in vivo, could prevent fetal brain injury and, specifically, neuronal injury.

Mouse model of intrauterine inflammation

CD-1 out-bred, timed pregnant mice (charles river laboratories, wilmington, MA) were utilized in an established model of inflammation-induced preterm birth. 23 , 26 as approximately 85% of spontaneous preterm births at less than 28 weeks are associated with intrauterine inflammation as demonstrated by the presence of histological chorioamnionitis, 27 this mouse model aptly mimics this common clinical scenario which occurs in many cases of spontaneous preterm birth.Fetal anoxia furthermore, using this model of preterm birth, we have demonstrated that prior to preterm delivery, exposure to intrauterine inflammation results in fetal brain injury and, thus, this model is also useful to assess interventions that might ameliorate or reduce adverse neonatal outcomes from inflammation-associated PTB. 23 – 25 survival surgery and intrauterine injections of lipopolysaccharide (LPS, 250 µg/dam in 100 µl PBS; from escherichia coli, 055:B5, sigma chemical co., st. Louis, MO) were performed on embryonic day 15 (E15) of gestation (term is 19 days) as previously reported. 26 briefly, anesthesia was obtained by a continuous flow of isofluorane/oxygen (air O 2), supplied by a mask that fits over the mouse’s head.Fetal anoxia after deep anesthesia was reached, a mini-laparotomy was performed in the lower abdomen. The right uterine horn was identified and LPS or saline was infused into the uterus between the first and the second gestational sacs. Routine closure was performed and the dams recovered in 3–5 minutes. Dams were humanely euthanized 6 hours after surgery by utilizing carbon dioxide (CO 2). Three dams were utilized per each treatment group. Immediately after euthanasia, four fetuses per dam were taken from lower uterine horns; as such, all fetuses from all dams were in the same proximity to where LPS was infused. Fetal brains were collected for message RNA (mrna) studies and for primary cortical neuronal cultures.Fetal anoxia guidelines for the care and use of animals were approved by the university of pennsylvania.

Treatment groups

After intrauterine (IU) infusion of LPS or NS (as previoulsy described) 23, dams were randomized to intraperitoneal (IP) treatment with mgso 4. The maternal mgso 4 injection protocol involved an IP dose of 270 mg/kg followed by 27 mg/kg every 20 minutes for 4 hours; injections were given in a volume of 0.1 ml. A second dose of 270 mg/kg was given at the end of the 4 hour period. An average dam weight at E15 is 40 grams. Control mice were injected with same volume of normal saline and at the same timing schedule. The selected protocol followed hallak et al. 28 , 29 A prior report, using this protocol in mice, demonstrated that 30 minutes after the injection of mgso 4, the magnesium values in the mothers’ blood samples were approximately double the normal values. 30 the use of this protocol in rats resulted in a 125% increase in magnesium level in the fetal forebrain after 4 hours. 31 this level of mgso 4 was demonstrated to prevent fetal brain damage associated with hypoxic-ischemic brain injury in a rodent model. 28 – 30 hence, using this protocol, the following 4 treatment groups were compared in these studies: 1) NS and NS (negative control); 2) LPS and NS (positive control); 3) LPS and mgso 4; and 4) NS and mgso 4.Fetal anoxia three dams from each treatment group were utilized for these experiments; from each dam, 4 fetal brains were used.

Primary cortical neuronal cultures

Using sterile technique, E15 fetal brains were harvested 4.5–6 hours after the IU randomization and placed into petri dishes containing cold ca ++/mg ++-free hanks balanced salt solution (HBSS; invitrogen, carlsbad, CA), ph 7.4. The cortex, a part of the fetal brain, was separated from meninges, olfactory bulbs, brain stem and cerebellum. Each cortex was minced, placed in 4 ml neurobasal medium (NBM; invitrogen, carlsbad, CA) containing 0.03% trypsin (invitrogen, carlsbad, CA) and incubated for 15 minutes at 37°C and 5% CO 2. Brain tissue was removed and placed in 4.5 ml NBM containing 10% fetal bovine serum (FBS) and allowed to settle to inactivate the trypsin.Fetal anoxia the medium was decanted and replaced with NBM supplemented with B-27 vitamin (invitrogen, carlsbad, CA) and 0.5mM L-glutamine and cells were dissociated by trituration. This media combination, NBM in the absence of fetal bovine serum, allows for the select growth of neurons and not glia (astrocytes or microgia). 32 , 33 cells were plated at low density (10 4 cells/ml) on poly-L-lysine (1 mg/ml; sigma-aldrich, st. Louis, MO) coated glass coverslips, using 12-well culture plates. Twelve fetal brains (n=12) from three dams (4 fetal brains per dam) per treatment group were utilized for the analysis of neuronal morphology per each treatment group. Cells were plated to equal density for each experiment.Fetal anoxia all experiments were performed in triplicate to assure the consistency of the results.

COMMENT

Prevention of neuronal injury in inflammation-associated PTB may be a key mechanism by which mgso 4 appears to be neuroprotective, specifically in decreasing CP in human studies. As we have previously demonstrated the presence of neuronal injury in fetal brains using a mouse model, 24 the principle findings of this study is that this neuronal injury can be ameliorated by antenatal administration of mgso 4.

PTB is enormous public health concern since many of these preterm infants survive with neurobehavioral, cognitive, and motor disabilities. 36 – 40 , 41 – 43 currently, the main theories regarding fetal brain injury and adverse neurological outcomes, including CP, from PTB, focus on specific structural findings of WMD. 44 – 46 however, clinically, these structural findings of WMD do not appear to explain the majority of observed neurological and neurobehavioral outcomes in ex-preterm children. 39 , 41 , 47 – 53 consequently, as adverse neurobehavioral phenotypes can occur in the absence of notable WMD, these findings call for a new paradigm regarding the pathogenesis of adverse neurobehavioral outcomes in ex-preterm children. 36 , 37 , 49 , 54 , 55 known mechanisms leading to these neurological/neurobehavioral outcomes in other disorders include neuronal abnormalities; specifically, abnormalities in synapses and dendritic arborization. 56 – 61 recent work from our laboratory supports the concept that neuronal injury may be an important mechanism for adverse neurological outcomes in ex-preterm children. 24 , 25 this current work, suggests that this neuronal injury can be abrogated by antenatal administration of mgso 4.Fetal anoxia if neuronal injury, with or without concomitant WMD, is a critical mechanism to long-term adverse neurobehavioral outcomes in ex-preterm children, then antenatal administration of mgso 4 may hold promise for preventing a spectrum of disorders in these children. However, it remains unknown the contribution of neuronal injury and/or WMD to each specific disorder observed in these offspring.

There are notable limitations to the study. The model used for these studies is not necessarily a specific rodent model of CP. This mouse model is intended to mimic the most common clinical scenario associated with preterm birth—that being one of intrauterine inflammation. As such, this model provides a method in which to test how intrauterine inflammation may affect fetal brain development and induce brain injury.Fetal anoxia

Our studies indicate that neuronal injury, at the time point chosen, can be prevented by mgso 4 even though the message expression of cytokines remained increased. A further limitation to our study is the assessment of only mrna expression and not protein levels of these specific cytokines. Yet, considering the short time interval to investigation, mrna expression is likely to be altered prior to protein changes. We recognize that assessment of both mrna and protein levels at longer intervals after exposure (from inflammation and mgso 4) may reveal different patterns of expression. Future work will be required to explore the interaction of cell death and immune mediators with neuronal injury.Fetal anoxia understanding these limitations, our results suggest that prevention of a cytokine response does not appear to be necessary to prevent neuronal injury nor is suppression of the fetal brain cytokine response the mechanism by which mgso 4 appears to prevent acute neuronal injury.

While a meta-analysis suggest that mgso 4 is not associated with neonatal mortality, 8 it has been proposed by others that mgso 4 may be implicated in increased fetal brain damage 14 and possibly neonatal mortality. 12 furthermore, animal studies have demonstrated that in higher doses mgso 4 can cause cell death. 14 therefore, we also evaluated the expression of the markers of cell death (caspases) in the whole fetal brains.Fetal anoxia as the expression of these genes was not altered with the administration of mgso 4, these studies do not support the concept that mgso 4 can induce cell death in the fetal brain – at least with the dosing regimen utilized for these studies. As these studies focused on the acute effects of mgso 4 on fetal brain, 4–6 hours after exposure to both inflammation and mgso 4, these studies can only report the acute effect of mgso 4on cell death.

These studies utilized CD-1 mice which are an out-bred strain of mice and hence provide a more diverse genetic background; thus, more aptly mimicking the human condition. However, the limitation of using an out-bred strain of mice is that there is variability in the maternal and fetal response to the same stimulus, such as LPS. 22 , 23 , 26 , 62 however, despite the variability of mrna expression, the findings of neuronal injury were consistent in fetal brains exposed to LPS as are the findings of mgso 4 preventing this injury.Fetal anoxia thus, we doubt that increasing the sample size (and hence animal utilization) will provide more insight into the pathogenesis of fetal brain injury and/or protection of injury by mgso 4. We believe that future work investigating long term outcomes after exposure to intrauterine inflammation in the presence or absence of mgso 4 is now warranted.

If neuronal injury is the main precursor to CP in ex-preterm children, then the ability of mgso 4 to prevent neuronal changes from inflammation may be a sufficient mechanism for decreasing these adverse outcomes. Furthermore, these studies suggest that mgso 4 may primarily be involved with protection at the level of neurons. What remains unclear is the contribution of WMD compared to neuronal injury for long-term outcomes.Fetal anoxia while WMD has been implicated in CP, 55 , 63 CP and other adverse neurobehavioral outcomes are known to occur in the absence of WMD. 36 , 37 , 49 , 54 , 55 animal work from our laboratory and others 23 , 44 , 45 , 64 have demonstrated that intrauterine inflammation can evoke WMD. Whether WMD or neuronal injury, or both are essential for adverse neurobehavioral outcomes in ex-preterm children is not yet clear. If WMD persists despite amelioration of neuronal injury, are offspring still at risk? Future work will need to address these important questions.

We acknowledge that there are difficulties in extrapolating findings in a rodent model to the clinical realm and this is one of the limitations of this study.Fetal anoxia however, elucidating the pathways involved in fetal brain injury from preterm birth in humans is not feasible. Therefore, for these types of studies, animal models of prenatal inflammation are generally used and provide a valuable insight into the mechanisms by which inflammation promotes fetal brain injury. Supporting our findings are data from other models of neuronal injury as well as the data involving mgso 4 use in other animal species. 28 – 30 mgso 4 has been investigated as a potential neuroprotective agent for glutametergic, HI and traumatic brain injury in the fetal and neonatal periods. 28 – 30 in one model of HI brain injury, maternal treatment with mgso 4 resulted in a significant fetal protection against moderate HI-induced brain damage. 28 – 30 although the mechanism of the initiation of HI fetal brain injury is distinct from that following the intrauterine inflammation, a common pathway of neuronal injury may serve to unify these injuries.Fetal anoxia despite animal and clinical studies evaluating the use of mgso 4 for neuroprotection, the precise mechanism by which mgso 4 serves to prevent neuronal injury is still under investigation. Several theories exist on possible mechanism by which mgso 4 prevents neuronal injury, which include: 1) acting as a non-competitive antagonist of NMDA-receptor, 2) preventing disruption of the blood-brain barrier permeability, and 3) inhibiting cell death. 15 – 19 , 66

Recognizing the work with mgso 4 in other models of prenatal/neonatal brain injury, our study is the first to date to investigate the use of mgso 4 in prenatal inflammation. Understanding limitations of animal models, these findings provide biological plausibility for the use of mgso 4 in clinical practice to prevent long-term adverse neurological outcomes.Fetal anoxia future work must address whether WMD, neuronal injury or both are required for the observed adverse neurobehavioral outcomes in ex-preterm infants as this may necessitate different interventional strategies. As the prevalence of adverse neurological outcomes in ex-preterm infants is increasing, understanding the mechanism of action of potential therapeutic interventions is critical if our goal is to decrease both acute and long term adverse outcomes for these children.