Hubert Monnerie

Inflammation-induced preterm birth alters neuronal morphology in the mouse fetal brain

Adverse neurological outcome is a major cause of long‐term morbidity in ex‐preterm children. To investigate the effect of parturition and inflammation on the fetal brain, we utilized two in vivo mouse models of preterm birth. To mimic the most common human scenario of preterm birth, we used a mouse model of intrauterine inflammation by intrauterine infusion of lipopolysaccharide (LPS). To investigate the effect of parturition on the immature fetal brain, in the absence of inflammation, we used a non‐infectious model of preterm birth by administering RU486. Pro‐inflammatory cytokines (IL‐10, IL‐1β, IL‐6 and TNF‐α) in amniotic fluid and inflammatory biomarkers in maternal serum and amniotic fluid were compared between the two models using ELISA. Pro‐inflammatory cytokine expression was evaluated in the whole fetal brains from the two models. Primary neuronal cultures from the fetal cortex were established from the different models and controls in order to compare the neuronal morphology. Only the intrauterine inflammation model resulted in an elevation of inflammatory biomarkers in the maternal serum and amniotic fluid. Exposure to inflammation‐induced preterm birth, but not non‐infectious preterm birth, also resulted in an increase in cytokine mRNA in whole fetal brain and in disrupted fetal neuronal morphology. In particular, Microtubule‐associated protein 2 (MAP2) staining was decreased and the number of dendrites was reduced (P < 0.001, ANOVA between groups). These results suggest that inflammation‐induced preterm birth and not the process of preterm birth may result in neuroinflammation and alter fetal neuronal morphology.

Beyond white matter damage: fetal neuronal injury in a mouse model of preterm birth

OBJECTIVE The purpose of this study was to elucidate possible mechanisms of fetal neuronal injury in inflammation-induced preterm birth. STUDY DESIGN With the use of a mouse model of preterm birth, the following primary cultures were prepared from fetal brains: (1) control neurons (CNs), (2) lipopolysaccharide-exposed neurons (LNs), (3) control coculture (CCC) that consisted of neurons and glia, and (4) lipopolysaccharide-exposed coculture (LCC) that consisted of lipopolysaccharide-exposed neurons and glia. CNs and LNs were treated with culture media from CN, LN, CCC, and LCC after 24 hours in vitro. Immunocytochemistry was performed for culture characterization and neuronal morphologic evidence. Quantitative polymerase chain reaction was performed for neuronal differentiation marker, microtubule-associated protein 2, and for cell death mediators, caspases 1, 3, and 9. RESULTS Lipopolysaccharide exposure in vivo did not influence neuronal or glial content in cocultures but decreased the expression of microtubule-associated protein 2 in LNs. Media from LNs and LCCs induced morphologic changes in control neurons that were comparable with LNs. The neuronal damage caused by in vivo exposure (LNs) could not be reversed by media from control groups. CONCLUSION Lipopolysaccharide-induced preterm birth may be responsible for irreversible neuronal injury.

Effect of excess extracellular glutamate on dendrite growth from cerebral cortical neurons at 3 days in vitro: Involvement of NMDA receptors

Glutamate is an important regulator of dendrite development; however, during cerebral ischemia, massive glutamate release can lead to neurodegeneration and death. An early consequence of glutamate excitotoxicity is dendrite injury, which often precedes cell death. We examined the effect of glutamate on dendrite growth from embryonic day 18 (E18) mouse cortical neurons grown for 3 days in vitro (DIV) and immunolabeled with anti‐microtubule‐associated protein (MAP)2 and anti‐neurofilament (NF)‐H, to identify dendrites and axons, respectively. Cortical neurons exposed to excess extracellular glutamate (100 μM) displayed reduced dendrite growth, which occurred in the absence of cell death. This effect was mimicked by the ionotropic glutamate receptor agonist N‐methyl‐D‐aspartate (NMDA) and blocked by the ionotropic glutamate receptor antagonist kynurenic acid and the NMDA receptor‐specific antagonist MK‐801. The non‐NMDA receptor agonist AMPA, however, did not affect process growth. Neither NMDA nor AMPA influenced neuron survival. Immunolabeling and Western blot analysis of NMDA receptors using antibodies against the NR1 subunit, demonstrated that immature cortical neurons used in this study, express NMDA receptors. These results suggest that excess glutamate decreases dendrite growth through a mechanism resulting from NMDA receptor subclass activation. Furthermore, these data support the possibility that excess glutamate activation of NMDA receptors mediate both cell death in mature neurons and the inhibitory effect of excess glutamate on dendrite growth in immature neurons or in the absence of cell death.

BMP-7 and excess glutamate: Opposing effects on dendrite growth from cerebral cortical neurons in vitro

Glutamate is an important regulator of dendrite development. During cerebral ischemia, however, there is massive release of glutamate reaching millimolar concentrations in the extracellular space. An early consequence of this excess glutamate is reduced dendrite growth. Bone morphogenetic protein-7 (BMP-7) a member of the transforming growth factor-beta (TGF-beta) superfamily has been demonstrated to enhance dendrite output from cerebral cortical and hippocampal neurons in vitro. However, it is not known whether BMP-7can prevent the reduced dendrite growth associated with excess glutamate or enhance dendrite growth after glutamate exposure. Therefore we quantified axon and primary, secondary, and total dendrite growth from embryonic mouse cortical neurons (E18) grown at low density in vitro in a chemically defined medium and exposed to glutamate (1 or 2 mM) for 48 h. Morphology and double immunolabeling (MAP2, NF-H) were used to identify cortical dendrites and axons after 3 DIV. In these short-term cultures, glutamate did not influence neuron survival. The addition of glutamate to cortical neurons, however, significantly attenuated dendrite output. This effect was mimicked by the addition of NMDA but not AMPA agonists and inhibited by the specific NMDA receptor antagonist MK-801. The reduction in dendrite growth mediated by excess glutamate was ameliorated by the administration of 30 or 100 ng/ml of BMP-7. In addition, when administered in a delayed fashion between 1 and 24 h after the initial glutamate exposure, BMP-7 was able to enhance dendrite growth, including primary dendrite number, primary dendrite length, and secondary dendritic branching. These findings demonstrate that BMP-7 can ameliorate reduced dendrite growth from cerebral cortical neurons associated with excess glutamate in vitro and are important because they may help explain why BMP-7 administration is associated with enhanced functional recovery in models of cerebral ischemia.

Dendritic alterations after dynamic axonal stretch injury in vitro.

Traumatic axonal injury (TAI) is the most common and important pathology of traumatic brain injury (TBI). However, little is known about potential indirect effects of TAI on dendrites. In this study, we used a well-established in vitro model of axonal stretch injury to investigate TAI-induced changes in dendrite morphology. Axons bridging two separated rat cortical neuron populations plated on a deformable substrate were used to create a zone of isolated stretch injury to axons. Following injury, we observed the formation of dendritic alterations or beading along the dendrite shaft. Dendritic beading formed within minutes after stretch then subsided over time. Pharmacological experiments revealed a sodium-dependent mechanism, while removing extracellular calcium exacerbated TAIs effect on dendrites. In addition, blocking ionotropic glutamate receptors with the N-methyl-d-aspartate (NMDA) receptor antagonist MK-801 prevented dendritic beading. These results demonstrate that axon mechanical injury directly affects dendrite morphology, highlighting an important bystander effect of TAI. The data also imply that TAI may alter dendrite structure and plasticity in vivo. An understanding of TAIs effect on dendrites is important since proper dendrite function is crucial for normal brain function and recovery after injury.

Reduced dendrite growth and altered glutamic acid decarboxylase (GAD) 65-and 67-kDa isoform protein expression from mouse cortical GABAergic neurons following excitotoxic injury in vitro

The vulnerability of brain cells to neurologic insults varies greatly, depending on their neuronal subpopulation. However, cells surviving pathological insults such as ischemia or brain trauma may undergo structural changes, e.g., altered process growth, that could compromise brain function. In this study, we examined the effect of glutamate excitotoxicity on dendrite growth from surviving cortical GABAergic neurons in vitro. Glutamate exposure did not affect GABAergic neuron viability, however, it significantly reduced dendrite growth from GABAergic neurons. This effect was blocked by the AMPA receptor antagonists NBQX and CFM-2, and mimicked by AMPA, but not NMDA. Glutamate excitotoxicity also caused an NMDA receptor-mediated decrease in the GABA synthesizing enzyme glutamic acid decarboxylase (GAD65/67) immunoreactivity from GABAergic neurons, measured using immunocytochemical and Western blot techniques. GAD is necessary for GABA synthesis; however, reduction of GABA by 3-mercaptopropionic acid (3-MPA), which inhibits GABA synthesis, did not alter dendrite growth. These results suggest that GABAergic cortical neurons are relatively resistant to excitotoxic-induced cell death, but they can display morphological and biochemical alterations which may impair their function.

Glutamate receptor agonist kainate enhances primary dendrite number and length from immature mouse cortical neurons in vitro

Glutamate is an important regulator of dendrite development that may inhibit, (during ischemic injury), or facilitate (during early development) dendrite growth. Previous studies have reported mainly on the N‐methyl‐D‐aspartate (NMDA) receptor‐mediated dendrite growth‐promoting effect of glutamate. In this study, we examined how the non‐NMDA receptor agonist kainate influenced dendrite growth. E18 mouse cortical neurons were grown for 3 days in vitro and immunolabeled with anti‐microtubule‐associated protein 2 (MAP2) and anti‐neurofilament (NF‐H), to identify dendrites and axons, respectively. Exposure of cortical neurons to kainate increased dendrite growth without affecting neuron survival. This effect was dose‐dependent, reversible and blocked by the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazoleproprionate (AMPA)/kainate receptor antagonist NBQX and the low‐affinity kainate receptor antagonist NS‐102, but not by the AMPA receptor antagonist CFM‐2. In addition, the NMDA receptor antagonist MK‐801 had no effect on kainate‐induced dendrite growth. Immunolabeling and Western blot analysis of kainate receptors using antibodies against the GluR6 and KA2 subunits, demonstrated that the immature cortical neurons used in this study express kainate receptor proteins. These results suggest that kainate‐induced non‐NMDA receptor activation promotes dendrite growth, and in particular primary dendrite number and length, from immature cortical neurons in vitro, and that kainate receptors may be directly involved in this process. Furthermore, these data support the possibility that like NMDA receptors, kainate receptor activation may also contribute to early neurite growth from cortical neurons in vitro.

Role of the NR2A/2B subunits of the N-methyl-D-aspartate receptor in glutamate-induced glutamic acid decarboxylase alteration in cortical GABAergic neurons in vitro.

The vulnerability of brain neuronal cell subpopulations to neurologic insults varies greatly. Among cells that survive a pathological insult, for example ischemia or brain trauma, some may undergo morphological and/or biochemical changes that may compromise brain function. The present study is a follow-up of our previous studies that investigated the effect of glutamate-induced excitotoxicity on the GABA synthesizing enzyme glutamic acid decarboxylase (GAD65/67)s expression in surviving DIV 11 cortical GABAergic neurons in vitro [Monnerie and Le Roux, (2007) Exp Neurol 205:367-382, (2008) Exp Neurol 213:145-153]. An N-methyl-D-aspartate receptor (NMDAR)-mediated decrease in GAD expression was found following glutamate exposure. Here we examined which NMDAR subtype(s) mediated the glutamate-induced change in GAD protein levels. Western blotting techniques on cortical neuron cultures showed that glutamates effect on GAD proteins was not altered by NR2B-containing diheteromeric (NR1/NR2B) receptor blockade. By contrast, blockade of triheteromeric (NR1/NR2A/NR2B) receptors fully protected against a decrease in GAD protein levels following glutamate exposure. When receptor location on the postsynaptic membrane was examined, extrasynaptic NMDAR stimulation was observed to be sufficient to decrease GAD protein levels similar to that observed after glutamate bath application. Blocking diheteromeric receptors prevented glutamates effect on GAD proteins after extrasynaptic NMDAR stimulation. Finally, NR2B subunit examination with site-specific antibodies demonstrated a glutamate-induced, calpain-mediated alteration in NR2B expression. These results suggest that glutamate-induced excitotoxic NMDAR stimulation in cultured GABAergic cortical neurons depends upon subunit composition and receptor location (synaptic vs. extrasynaptic) on the neuronal membrane. Biochemical alterations in surviving cortical GABAergic neurons in various disease states may contribute to the altered balance between excitation and inhibition that is often observed after injury.

β-Amyloid-induced reactive astrocytes display altered ability to support dendrite and axon growth from mouse cerebral cortical neurons in vitro

Abstract Objectives: The presence of β-amyloid (βA) deposition, induction of reactive gliosis and dystrophic neurites, is a characteristic feature of neuritic plaques in Alzheimers disease. In vitro, βA-exposed astrocytes become reactive, similar to astrocytes in contact with βA plaques in vivo. How βA-exposed reactive astrocytes support neuron process growth, however, is not well defined. Therefore, we used neuron/astrocyte co-cultures in which astrocytes had been grown on βA, to assess whether process growth was altered. Methods: Purified rat cortical astrocytes were plated on the βA peptides neurotoxic fragment (25–35), the scrambled (35–25) peptide, or poly-D-lysine alone and grown to confluency before mouse cortical neurons were seeded at low density onto the astrocyte monolayer. Cell survival was assessed using trypan blue, lactate dehydrogenase release and propidium iodide. Process growth was analyzed using specific antibodies against MAP2 and the 200 kDa neurofilament subunit (NF-H) to identify dendrites and axons, respectively. Results: βA-exposed astrocytes changed dramatically from their flat polygonal shape into stellate process-bearing morphology. Viability however, was not affected. Immunocytochemical analysis of neuronal processes using anti-MAP2 and anti-NF-H, demonstrated that βA (25–35)induced reactive astrocytes had an altered ability to support dendrite and axon growth after 3 days in vitro. Indeed, primary dendrite number and axon length were decreased by 30 and 26%, respectively, compared with control astrocytes, whereas individual primary dendrite length increased by 20%. Astrocyte support of dendritic branching, however, was not affected by βA. Discussion: We conclude that an astrocyte reaction to βA may contribute, in part, to neuronal dystrophy associated with βA plaques.

Growth of rat cortical neurons on DuraGen, a collagen-based dural graft matrix

Abstract Objectives: DuraGen, a collagen-based dural graft matrix, is frequently used in clinical neurosurgery. In the present study we examined whether DuraGen influenced neuron survival of or process growth from cerebral cortex neurons in culture. Methods: Dissociated E19 rat cerebral cortical neurons were cultured at low density on poly-L-lysine or on cryostat-sectioned DuraGen. Neuron survival was assessed using morphological criteria, fluorescein diacetate (FDA) and propidium iodide (PI), nuclear staining and TUNEL labeling. Process growth was analysed using specific antibodies against MAP2 and the 200 kDa neurofilament subunit (NF-H) to identify dendrites and axons, respectively. Results: In immature cultures (3 days in vitro, DIV), nearly 70% of the neurons remained viable in control and DuraGen-exposed cells. In mature cultures (10 DIV), ∼45% of the neurons were viable. Survival was similar in DuraGen cultures and controls. Cell viability also was similar when DuraGen conditioned the medium, but was not in contact with the neurons. When 10-day-old cultures were treated with glutamate (100 μmol/l for 24 hours) to elicit excitotoxic injury, a 40% decrease in neuron survival was observed. DuraGens presence neither exacerbated nor attenuated glutamate-induced excitotoxic neuron death. The amount of necrotic or apoptotic cells also was similar in control and DuraGen cultures. Finally, DuraGen had an equal ability to support both axon and dendrite growth as poly-L-lysine. Conclusion: Our findings demonstrate that DuraGen has no adverse effect on survival of or process growth from cerebral cortical neurons in vitro. These data support DuraGens biosafety as a dural substitute in clinical neurosurgery.