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Modulation of Triggering receptor expressed in myeloid cells 2 by gene transfer as novel neuroprotective estrategy for neonatal hypoxic ischemic brain injury using behavioural outcome as readout

Final Report Summary - NEONATAL HI INJURY (Modulation of Triggering receptor expressed in myeloid cells 2 by gene transfer as novel neuroprotective estrategy for neonatal hypoxic ischemic brain injury using behavioural outcome as readout)

Insults during the perinatal stage of brain development lead to major causes of neurological disability throughout life, ranging from motor deficits, cognitive limitations, learning difficulties and even severe disabilities, such as cerebral palsy. In term newborn infants, hypoxic/ischemic (HI) brain injury is the most common cause of encephalopathy and seizures. Despite major improvements in neonatal care, there are no established therapeutic procedures successful for the prevention or treatment of perinatal brain lesions, although hypothermia is an alternative for moderate cases. Understanding the evolution of neonatal hypoxic/ischemic is essential for novel neuroprotective approaches.
In contrast to the adult, the immature brain displays distinct physiological and morphological features as a consequence of its ongoing postnatal development; however, the neonatal brain damage is poorly characterized. Furthermore, experimental evidences also suggest that the inflammatory response associated to a CNS injury is exacerbated during immaturity. As brain development substantially influences the progression and hallmarks of brain injury, it is not possible to apply the results of medical research obtained in adults reliably to babies.
The field of endogenous regulatory receptors modulating inflammatory cell activation is largely unknown in the brain and no studies have yet focused on the damaged neonatal brain. In consequence, reaching a detailed knowledge of the endogenous inflammatory regulation the CNS during the early postnatal stage is essential for the proper development of neuroprotective therapies specific for this period. For this reason, this project aims to explore the efficacy of the endogenous regulatory receptors during postnatal development and in the control of neuroinflammation in the newborn brain, mainly on TREM2 (triggering receptor expressed on myeloid cells 2) in the control of neuroinflammation following HI in the newborn brain, taking into account the behavioral outcome.

TREM2 expression during normal development
Microglia, the resident innate immune cells of the central nervous system, are constantly monitoring the brain parenchyma, cleaning the cell debris or the synaptic contacts overproduced during postnatal development and also maintaining the brain homeostasis. In this context, the postnatal microglia need some control over the innate immune response. We first characterized the distribution of TREM2 protein in mice brain during postnatal development, from Postnatal day (P) 1 to 14 by immunostaining. In our study, TREM2 protein was expressed only in microglia/macrophages and is developmentally downregulated in a region-dependent manner. Its expression persisted in white matter, mainly in caudal corpus callosum, and the neurogenic subventricular zone for a longer time than in grey matter. Additionally, the phenotypes of the TREM2+ microglia differ; expressing antigen presentation markers (CD16/32, MHCII and CD86) and phagocytic marker (CD68) in different regions as well as with different intensity till P7. The mannose receptor (CD206) colocalized with TREM2 only at P1-P3 in the subventricular zone and cingulum, while others persisted at low intensities till P7.
In conclusion, the spatiotemporal expression pattern and characterization of TREM2 suggest its plausible roles in phagocytosis, progenitor’s fate determination or microglia phenotype modulation during postnatal development. The temporal and regional modulation of TREM2 might give us the basis for a better understanding of mechanisms involved in neurodegenerative disorders following perinatal brain injury.

Short- and Long-term Inflammatory response in neonatal brain after Hypoxic ischemic lesion
At cellular level: In order to understand how inflammation progress after HI lesion at birth we characterize in detail the neuropathology and glial/inflammatory response, from 3 hours to 100 days, after carotid occlusion and hypoxia (8% O2, 55 minutes) to the C57/BL6 P7 mouse, a model of systematic asphyxia occurring at birth. Massive tissue injury and atrophy in the ipsilateral hippocampus, corpus callosum and caudate-putamen is consistently shown. Astrogliosis peaks at 14 days, but glial scar is still evident at day 100. Microgliosis peaks at 3-7 days and decreases by day 14. Neutrophils increase in the ventricles and hippocampal vasculature, showing also parenchymal extravasation at 7 days. Remarkably, delayed milder atrophy is also seen in the contralateral hippocampus and corpus callosum, areas showing astrogliosis and microgliosis during the first 72 hours.

At molecular level: We also examined the signaling pathways involved and the effector molecules. The signal transduction and activator of transcription factor 3 (STAT3) is known to modulate injury following imbalance between pro- and anti-inflammatory cytokines in peripheral and central nervous system injury making it a potential molecule for study. Hence we investigates the temporal expression of Interleukin (IL)-6, IL-1beta, TNFalpha, IL-1ra, IL-4, IL-10, IL-13 and pSTAT3 after HI in P7 mice. Protein array illustrated notable changes in cytokines expressed in both the hemispheres in a time dependent manner. The major pro-inflammatory cytokines showing immediate changes between ipsi- and contralateral hemispheres were IL-6 and IL-1beta. The anti-inflammatory cytokines, IL-4 and IL-13 demonstrated a delayed augmentation with no prominent differences between hemispheres, while IL-1ra showed two distinct peaks of expression spread over time. We also illustrate for the first time the spatio-temporal activation of pSTAT3 after a neonatal HI in mice brain. The main regions expressing pSTAT3 were hippocampus and corpus callosum. pSTAT3+ cells were mostly a subpopulation of activated astrocytes (GFAP+) and microglia/macrophages (F4/80+) seen only in the ipsilateral hemisphere at all the time-points studied. The highest expression of pSTAT3+ cells was observed to be around 24-48 hours in astrocytes and microglia/macrophages. In conclusion, our study highlights a synchronized expression of some pro- and anti-inflammatory cytokines, especially in long-term not previously defined. It also points towards a significant role of STAT3 signalling following micro- and astrogliosis in the physiopathology of neonatal HI related brain injury. In the study, a shift from pro-inflammatory to anti-inflammatory cytokine profile was also noted as the injury progressed. We suggest that while designing efficient neuroprotective therapies using inflammatory molecules, the time of intervention and balance between the pro- and anti- inflammatory cytokines must be considered.

At cognitive level: In a preliminary experiment, we analyzed the behavioral profile of C7BL6/C mice following neonatal hypoxia-ischemia. The results indicated that H/I affected motor activity, behavioral and psychological symptoms and cognitive function. The results also provided suggested the existence of a gender component that would be interesting to be studied.

TREM2 Expression after HI
We previously describe TREM2 expression only in the subcortical white matter and in the cingulum at Postnatal 7 and 10. Following HI, an increase in TREM2 staining was observed in the subcortical white matter, hippocampus and in the caudate-putamen, cortex and thalamus in the IL hemisphere, starting after 12-24hours till 7 days post hypoxia. This are the regions were microgliosis and astrogliosis were observed. Additionally, the Contralateral hippocampus and corpus callosum have also shown TREM2 overexpression at early time points, where milder atrophy was observed. These results suggest that the TREM2 might be involved in the process of the tissue damage after hypoxia ischemia in neonatal mice.
The characterization of the population of cells overexpressing TREM2 after lesion is under progress.
This detailed and long-term cellular response characterization of the ipsilateral and contralateral hemisphere after HI may help in the design of better therapeutic strategies.

This project was performed in collaboration with Kalpana Shrivastava, Gemma Llovera, Mireia Recasens, Aida Muntsant, Virginia Torres-Lista and Berta González.

Socio-economic impact
At present, optimal management of an HI brain injury involves prompt resuscitation, careful supportive care, including prevention of hyperthermia and hypoglycaemia, and treatment of clinical and frequent or prolonged subclinical seizures. Although hypothermia is a promising new therapy, and recent studies suggests that head or whole-body cooling administered within 6 hours of birth reduces the incidence of death or moderate/severe disability at 12 to 22 months, there is compelling need for well-designed clinical trials to address the treatment of ongoing brain injury in the setting of hypoxia-ischemia and seizures. Prevention of perinatal brain damage is of major importance for public health, families and individuals. As medical progress has reduced infant deaths considerably in the last decades, the absolute number of neurological handicaps of perinatal origin is rising in Western countries due to increasing survival of preterm infants. Of the approximate four million live births per year within the EU, approximately 60.000 infants are born each year with some sort of brain injury.
The physiopathology of neonatal brain damage is poorly characterized, in this sense, evidences from studies performed in several laboratories in the last few decades have shown that the production of inflammatory molecules and inducers of oxidative stress by inflammatory cell types contributes to extension of neuronal damage and tissue injury induced by acute or chronic neurodegeneration. This is one of the mayor contributions of this project.
Advances in the knowledge of the endogenous regulation of the neuroinflammatory response are essential for the development of therapeutic strategies that will open new opportunities to prevent the progression of neuronal damage, even promoting tissue regeneration. This field requires a detailed and meticulous study of endogenous mechanisms controlling and modulating the brain inflammatory response but also in normal brain development, this is another of the contributions of the project.