Ana González-Manteiga

Myelomeningocele (MMC) is a congenital birth defect of the spine and spinal cord, commonly treated clinically through prenatal or postnatal surgery by repairing the unclosed spinal canal. Having previously developed a PLA/PCL polymer smart patch for this condition, we aim to further expand the potential therapeutic options by providing additional cellular and biochemical support in addition to its mechanical properties. Bone morphogenetic proteins (BMPs) are a large class of secreted factors that serve as modulators of development in multiple organ systems, including the CNS. We hypothesize that our smart patch mitigates the astrogenesis induced, at least partly, by increased BMP activity during MMC. To test this hypothesis, neural stem or precursor cells were isolated from rat fetuses and cultured in the presence of Noggin, an endogenous antagonist of BMP action, with recombinant BMPs. We found that the developed PLA/PCL patch not only serves as a biocompatible material for developing neural stem cells but was also able to act as a carrier for BMP–Notch pathway inhibitor Noggin, effectively minimizing the effect of BMP2 or BMP4 on NPCs cultured with the Noggin-loaded patch.

Brain damage is the major cause of permanent disability and it is particularly relevant in the elderly. While most studies focused on the immediate phase of neuronal loss upon injury, much less is known about the process of axonal regeneration after damage. The development of new refined preclinical models to investigate neuronal regeneration and the recovery of brain tissue upon injury is a major unmet challenge. Here, we present a novel experimental paradigm in mice that entails the (i) tracing of cortico-callosal connections, (ii) a mechanical lesion of the motor cortex, (iii) the stereological and histological analysis of the damaged tissue, and (iv) the functional characterization of motor deficits. By combining conventional microscopy with semi-automated 3D reconstruction, this approach allows the analysis of fine subcellular structures, such as axonal terminals, with the tridimensional overview of the connectivity and tissue integrity around the lesioned area. Since this 3D reconstruction is performed in serial sections, multiple labeling can be performed by combining diverse histological markers. We provide an example of how this methodology can be used to study cellular interactions. Namely, we show the correlation between active microglial cells and the perineuronal nets that envelop parvalbumin interneurons. In conclusion, this novel experimental paradigm will contribute to a better understanding of the molecular and cellular interactions underpinning the process of cortical regeneration upon brain damage.

Hyperammonemia induces neuroinflammation and increases GABAergic tone in the cerebellum which contributes to cognitive and motor impairment in hepatic encephalopathy (HE). The link between neuroinflammation and GABAergic tone remains unknown. New treatments reducing neuroinflammation and GABAergic tone could improve neurological impairment. The aims were, in hyperammonemic rats, to assess whether: We analyzed in control and hyperammonemic rats, treated or not with sulforaphane, (a) learning in the Y maze; (b) motor coordination in the beam walking; (c) glutamate-NO-cGMP pathway and extracellular GABA by microdialysis; (d) microglial activation, by analyzing by immunohistochemistry or Western blot markers of pro-inflammatory (M1) (IL-1b, Iba-1) and anti-inflammatory (M2) microglia (Iba1, IL-4, IL-10, Arg1, YM-1); and (e) membrane expression of the GABA transporter GAT-3. Hyperammonemia induces activation of astrocytes and microglia in the cerebellum as assessed by immunohistochemistry. Hyperammonemia-induced neuroinflammation is associated with increased membrane expression of the GABA transporter GAT-3, mainly in activated astrocytes. This is also associated with increased extracellular GABA in the cerebellum and with motor in-coordination and impaired learning ability in the Y maze. Sulforaphane promotes polarization of microglia from the M1 to the M2 phenotype, reducing IL-1b and increasing IL-4, IL-10, Arg1, and YM-1 in the cerebellum. This is associated with astrocytes deactivation and normalization of GAT-3 membrane expression, extracellular GABA, glutamate-nitric oxide-cGMP pathway, and learning and motor coordination. Neuroinflammation increases GABAergic tone in the cerebellum by increasing GAT-3 membrane expression. This impairs motor coordination and learning in the Y maze. Sulforaphane could be a new therapeutic approach to improve cognitive and motor function in hyperammonemia, hepatic encephalopathy, and other pathologies associated with neuroinflammation by promoting microglia differentiation from M1 to M2.