Neuronal injury in inflammation-induced preterm birth may be a critical mechanism for adverse neurological outcomes in the ex-preterm children. In previous work in our laboratory, we have demonstrated the presence of neuronal injury in preterm fetal brains using an LPS-exposed mouse model. 16
These current studies now demonstrated that this neuronal injury can be propagated by the injured neurons. This suggests that while the trigger of inflammatory pathways may be ‘removed’ after the preterm birth that neuronal injury may continue into the neonatal period and beyond, with the presence of injured neurons. If valid in vivo
, this finding may imply that a chronic neuronal injury may be present in the brains of ex-preterm neonates. These findings and their implications mandate a paradigm shift in the conceptualization of fetal brain injury in a preterm birth.
A number of animal models have been developed to elucidate the mechanisms of inflammation induced brain damage.20, 27-31
While all animal models have inherent limitations, these studies and such mechanistic questions cannot be assessed in humans. It is noted that CNS development in the rodent differs from the human in regards to oligodendrocytes development. In the E15 fetal mouse brain, myelination is not completed. While pro-oligodendrocytes are present, mature oligodendrocytes are not. We have previously demonstrated that intrauterine inflammation results in a loss of these pro-oligodendrocytes. 20
Notably, for these studies, we are interested in neuron-neuron and neuron-glial interactions and hence the lack of mature oligodendrocytes for these studies is not a limitation. The strengths of the study are that this model utilizes an in utero
or local model which is believed to mirror the inflammation associated with human preterm birth. 18
Furthermore, the molecular events revealed in the studies most likely represent what occurs in vivo
in the fetal brain with intact paracrine effects of neuron-neuron and glial-neuronal mechanisms.
A potential limitation of the study; if only as an unknown factor, is the utilization of CO2 for euthanasia. The possible consequences of rising CO2 on health of an embryo are well realized. However, since both control and LPS-exposed groups were euthanized with the same amount of CO2, the effect would be equal across treatment groups and hence is unlikely to be the reason neuronal injury was observed
Animal models, used to elucidate the mechanisms by which preterm birth promotes fetal brain injury,27,28,31
have, to date, concentrated on WMD and astrogliosis as the outcomes of interest. However, recent studies suggest that adverse neurological outcomes in ex-preterm infants encompass many disorders beyond CP 4-11
and neuronal injury is known to mechanistically contribute to such deficits as found in other diseases involving neurotoxicity.32,33
As demonstrated from our laboratory, an intrauterine LPS induces cytokine production in the fetal brain20
and subsequent fetal neuronal injury.13
This study provides further understanding into the mechanisms promoting the neuronal injury.
Neuroinflammation and neurotoxicity are frequently considered distinct but common hallmarks of several neuroinflammatory disorders, including Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, and Alzheimer's disease. Neuron-neuron and glial-neuronal interactions have been demonstrated to be critical mechanisms in these disease states.14
Microglial cells, the brain resident macrophages, and astrocytes, the most prevalent type of cell in brain, are actively involved in the control of neuronal activities both in fetus and adult.34
At the same time, neurons influence glial and neuronal functions, through direct cell-to-cell interactions as well as the release of soluble mediators.35
Similarly, these studies herein demonstrate that neurons participate in cross talk in fetal brain injury in the setting of intrauterine inflammation. Most importantly, this study has demonstrated that neurons injured during fetal life are sufficient to cause further neuronal injury.
This work suggests that mechanisms of injury in fetal brain damage in preterm birth are similar to other neuroinflammatory disorders and that neuron-neuron communication are implicated in the observed injury. Further work is required to determine the specific contribution of glia, specifically astrocytes, to fetal brain injury. It could be speculated, based on our observation of similar MAP2 expression in control and LPS-exposed co-cultures, as opposed to a differential expression in neuronal cultures, that glia (present in co-culture) may serve a neuroprotective role through paracrine interactions.
It appears self-evident that alterations in neuronal morphology, as we have now demonstrated, is present in fetal brains exposed to intrauterine inflammation, is detrimental for neuronal function in that the presence of fewer synaptic spines implies a diminished neuronal information processing.36
Dendritic morphology has been demonstrated to be vital to normal neuronal information processing and allows the neuron to receive messages and permits cortical connectivity.37
A decreased MAP2 staining in cultured neurons is demonstrated to be associated with fragility and be a result of excitotoxicity.38, 39
Our findings, which demonstrate that inflammation-induced preterm birth results in diminished MAP2 staining and MAP2 expression, suggest that intrauterine inflammation may result in abnormal neuronal processing and synaptic communication. Our results further indicate that the neuronal injury appears to be present in inflammation-induced preterm birth, accompanies WMD, 20
and may explain the adverse neurological outcomes, including cognitive and behavioral deficits.
In these studies, the observed phenomenon that injured neurons alone are sufficient to induce the neuronal injury in previously unaffected neurons implies that local paracrine effects are important in are important in development of a global brain injury and propagation of neuronal morphological change. These neuron-neuron interactions have immense implications for human outcomes. An important finding in our study is that the co-culture characterization did not reveal any changes in percentage of glia or neurons nor did we observe an increase in Caspase expression, suggesting that astrogliosis and neuronal death may be later findings in neurotoxicity. The inability of the normal neuronal or glial-neuronal milieu (from control cortical or co-cultures) to reverse the neuronal injury suggests the irreversibility of neuronal injury possibly due to excitotoxic pathways. Alternatively, this may be a finding limited to an ex vivo study and, in fact, neuronal injury may be reversible in vivo as neuroprotective mechanisms may require paracrine effects which are present in the intact fetal brain.
Consequently, these results have immediate consequences both for research and for the development of novel therapies. As there are no proposed mechanisms of neuronal injury in the setting of preterm birth, these data are crucial for directing new research directions. With the increasing prevalence of adverse neurological outcomes in ex-preterm infants, understanding the mechanism of fetal brain injury in a preterm birth is critical if we are to decrease both acute and long term adverse outcomes for these children. Based on our recent work, targeting neuronal injury warrants active investigations. Specifically, future studies are required to determine if the neuronal-neuronal injury continues into the postnatal period or whether there are innate neuroprotective mechanisms, such as neurotrophins, that limit injury. If neuronal-neuronal injury is a perpetuating source of brain injury in the ex-preterm infant, these events become critical therapeutic targets.