Modifying the Secretome of Mesenchymal Stem Cells Prolongs the Regenerative Treatment Window for Encephalopathy of Prematurity.

Clinical treatment options to combat Encephalopathy of Prematurity (EoP) are still lacking. We, and others, have proposed (intranasal) mesenchymal stem cells (MSCs) as a potent therapeutic strategy to boost white matter repair in the injured preterm brain. Using a double-hit mouse model of diffuse white matter injury, we previously showed that the efficacy of MSC treatment was time dependent, with a significant decrease in functional and histological improvements after the postponement of cell administration. In this follow-up study, we aimed to investigate the mechanisms underlying this loss of therapeutic efficacy. Additionally, we optimized the regenerative potential of MSCs by means of genetic engineering with the transient hypersecretion of beneficial factors, in order to prolong the treatment window. Though the cerebral expression of known chemoattractants was stable over time, the migration of MSCs to the injured brain was partially impaired. Moreover, using a primary oligodendrocyte (OL) culture, we showed that the rescue of injured OLs was reduced after delayed MSC coculture. Cocultures of modified MSCs, hypersecreting IGF1, LIF, IL11, or IL10, with primary microglia and OLs, revealed a superior treatment efficacy over naïve MSCs. Additionally, we showed that the delayed intranasal administration of IGF1-, LIF-, or IL11-hypersecreting MSCs, improved myelination and the functional outcome in EoP mice. In conclusion, the impaired migration and regenerative capacity of intranasally applied MSCs likely underlie the observed loss of efficacy after delayed treatment. The intranasal administration of IGF1-, LIF-, or IL11-hypersecreting MSCs, is a promising optimization strategy to prolong the window for effective MSC treatment in preterm infants with EoP.

Early predictors of neurodevelopment after perinatal arterial ischemic stroke: a systematic review and meta-analysis.

BACKGROUND AND AIMS: Perinatal arterial ischemic stroke (PAIS) often has lifelong neurodevelopmental consequences. We aimed to review early predictors (<4 months of age) of long-term outcome. METHODS: We carried out a systematic literature search (PubMed and Embase), and included articles describing term-born infants with PAIS that underwent a diagnostic procedure within four months of age, and had any reported outcome parameter ≥12 months of age. Two independent reviewers included studies and performed risk of bias analysis. RESULTS: We included 41 articles reporting on 1395 infants, whereof 1255 (90%) infants underwent follow-up at a median of 4 years. A meta-analysis was performed for the development of cerebral palsy (n = 23 studies); the best predictor was the qualitative or quantitative assessment of the corticospinal tracts on MRI, followed by standardized motor assessments. For long-term cognitive functioning, bedside techniques including (a)EEG and NIRS might be valuable. Injury to the optic radiation on DTI correctly predicted visual field defects. No predictors could be identified for behavior, language, and post-neonatal epilepsy. CONCLUSION: Corticospinal tract assessment on MRI and standardized motor assessments are best to predict cerebral palsy after PAIS. Future research should be focused on improving outcome prediction for non-motor outcomes. IMPACT: We present a systematic review of early predictors for various long-term outcome categories after perinatal arterial ischemic stroke (PAIS), including a meta-analysis for the outcome unilateral spastic cerebral palsy. Corticospinal tract assessment on MRI and standardized motor assessments are best to predict cerebral palsy after PAIS, while bedside techniques such as (a)EEG and NIRS might improve cognitive outcome prediction. Future research should be focused on improving outcome prediction for non-motor outcomes.

The impact of trophic and immunomodulatory factors on oligodendrocyte maturation: Potential treatments for encephalopathy of prematurity.

Encephalopathy of prematurity (EoP) is a major cause of morbidity in preterm neonates, causing neurodevelopmental adversities that can lead to lifelong impairments. Preterm birth-related insults, such as cerebral oxygen fluctuations and perinatal inflammation, are believed to negatively impact brain development, leading to a range of brain abnormalities. Diffuse white matter injury is a major hallmark of EoP and characterized by widespread hypomyelination, the result of disturbances in oligodendrocyte lineage development. At present, there are no treatment options available, despite the enormous burden of EoP on patients, their families, and society. Over the years, research in the field of neonatal brain injury and other white matter pathologies has led to the identification of several promising trophic factors and cytokines that contribute to the survival and maturation of oligodendrocytes, and/or dampening neuroinflammation. In this review, we discuss the current literature on selected factors and their therapeutic potential to combat EoP, covering a wide range of in vitro, preclinical and clinical studies. Furthermore, we offer a future perspective on the translatability of these factors into clinical practice.

Intranasal mesenchymal stem cell therapy to boost myelination after encephalopathy of prematurity.

Encephalopathy of prematurity (EoP) is a common cause of long-term neurodevelopmental morbidity in extreme preterm infants. Diffuse white matter injury (dWMI) is currently the most commonly observed form of EoP. Impaired maturation of oligodendrocytes (OLs) is the main underlying pathophysiological mechanism. No therapies are currently available to combat dWMI. Intranasal application of mesenchymal stem cells (MSCs) is a promising therapeutic option to boost neuroregeneration after injury. Here, we developed a double-hit dWMI mouse model and investigated the therapeutic potential of intranasal MSC therapy. Postnatal systemic inflammation and hypoxia-ischemia led to transient deficits in cortical myelination and OL maturation, functional deficits and neuroinflammation. Intranasal MSCs migrated dispersedly into the injured brain and potently improved myelination and functional outcome, dampened cerebral inflammationand rescued OL maturation after dWMI. Cocultures of MSCs with primary microglia or OLs show that MSCs secrete factors that directly promote OL maturation and dampen neuroinflammation. We show that MSCs adapt their secretome after ex vivo exposure to dWMI milieu and identified several factors including IGF1, EGF, LIF, and IL11 that potently boost OL maturation. Additionally, we showed that MSC-treated dWMI brains express different levels of these beneficial secreted factors. In conclusion, the combination of postnatal systemic inflammation and hypoxia-ischemia leads to a pattern of developmental brain abnormalities that mimics the clinical situation. Intranasal delivery of MSCs, that secrete several beneficial factors in situ, is a promising strategy to restore myelination after dWMI and subsequently improve the neurodevelopmental outcome of extreme preterm infants in the future.

Neuroprotection offered by mesenchymal stem cells in perinatal brain injury: Role of mitochondria, inflammation, and reactive oxygen species.

Preclinical studies have shown that mesenchymal stem cells have a positive effect in perinatal brain injury models. The mechanisms that cause these neurotherapeutic effects are not entirely intelligible. Mitochondrial damage, inflammation, and reactive oxygen species are considered to be critically involved in the development of injury. Mesenchymal stem cells have immunomodulatory action and exert mitoprotective effects which attenuate production of reactive oxygen species and promote restoration of tissue function and metabolism after perinatal insults. This review summarizes the present state, the underlying causes, challenges and possibilities for effective clinical translation of mesenchymal stem cell therapy.

Nasal administration of mesenchymal stem cells reverses chemotherapy-induced peripheral neuropathy in mice.

Chemotherapy-induced peripheral neuropathy (CIPN) is one of the most frequently reported adverse effects of cancer treatment. CIPN often persists long after treatment completion and has detrimental effects on patient's quality of life. There are no efficacious FDA-approved drugs for CIPN. We recently demonstrated that nasal administration of mesenchymal stem cells (MSC) reverses the cognitive deficits induced by cisplatin in mice. Here we show that nasal administration of MSC after cisplatin- or paclitaxel treatment- completely reverses signs of established CIPN, including mechanical allodynia, spontaneous pain, and loss of intraepidermal nerve fibers (IENF) in the paw. The resolution of CIPN is associated with normalization of the cisplatin-induced decrease in mitochondrial bioenergetics in DRG neurons. Nasally administered MSC enter rapidly the meninges of the brain, spinal cord and peripheral lymph nodes to promote IL-10 production by macrophages. MSC-mediated resolution of mechanical allodynia, recovery of IENFs and restoration of DRG mitochondrial function critically depends on IL-10 production. MSC from IL-10 knockout animals are not capable of reversing the symptoms of CIPN. Moreover, WT MSC do not reverse CIPN in mice lacking IL-10 receptors on peripheral sensory neurons. In conclusion, only two nasal administrations of MSC fully reverse CIPN and the associated mitochondrial abnormalities via an IL-10 dependent pathway. Since MSC are already applied clinically, we propose that nasal MSC treatment could become a powerful treatment for the large group of patients suffering from neurotoxicities of cancer treatment.

Cerebellar injury in term neonates with hypoxic-ischemic encephalopathy is underestimated.

BACKGROUND: Postmortem examinations frequently show cerebellar injury in infants with severe hypoxic-ischemic encephalopathy (HIE), while it is less well visible on MRI. The primary aim was to investigate the correlation between cerebellar apparent diffusion coefficient (ADC) values and histopathology in infants with HIE. The secondary aim was to compare ADC values in the cerebellum of infants with HIE and infants without brain injury. METHODS: ADC values in the cerebellar vermis, hemispheres and dentate nucleus (DN) of (near-)term infants with HIE (n = 33) within the first week after birth were compared with neonates with congenital non-cardiac anomalies, normal postoperative MRIs and normal outcome (n = 22). Microglia/macrophage activation was assessed using CD68 and/or HLA-DR staining and Purkinje cell (PC) injury using H&E-stained slices. The correlation between ADC values and the histopathological measures was analyzed. RESULTS: ADC values in the vermis (p = 0.021) and DN (p < 0.001) were significantly lower in infants with HIE compared to controls. ADC values in the cerebellar hemispheres were comparable. ADC values in the vermis were correlated with the number and percentage of normal PCs; otherwise ADC values and histology were not correlated. CONCLUSION: Histopathological injury in the cerebellum is common in infants with HIE. ADC values underestimate histopathological injury. IMPACT: ADC values might underestimate cerebellar injury in neonates with HIE. ADC values in the vermis and dentate nucleus of infants with HIE are lower compared to controls, but not in the cerebellar hemispheres. Abnormal ADC values are only found when cytotoxic edema is very severe. ADC values in the vermis are correlated with Purkinje cell injury in the vermis; furthermore, there were no correlations between ADC values and histopathological measures.

Regenerative Therapies to Restore Interneuron Disturbances in Experimental Models of Encephalopathy of Prematurity.

Encephalopathy of Prematurity (EoP) is a major cause of morbidity in (extreme) preterm neonates. Though the majority of EoP research has focused on failure of oligodendrocyte maturation as an underlying pathophysiological mechanism, recent pioneer work has identified developmental disturbances in inhibitory interneurons to contribute to EoP. Here we investigated interneuron abnormalities in two experimental models of EoP and explored the potential of two promising treatment strategies, namely intranasal mesenchymal stem cells (MSCs) or insulin-like growth factor I (IGF1), to restore interneuron development. In rats, fetal inflammation and postnatal hypoxia led to a transient increase in total cortical interneuron numbers, with a layer-specific deficit in parvalbumin (PV)+ interneurons. Additionally, a transient excess of total cortical cell density was observed, including excitatory neuron numbers. In the hippocampal cornu ammonis (CA) 1 region, long-term deficits in total interneuron numbers and PV+ subtype were observed. In mice subjected to postnatal hypoxia/ischemia and systemic inflammation, total numbers of cortical interneurons remained unaffected; however, subtype analysis revealed a global, transient reduction in PV+ cells and a long-lasting layer-specific increase in vasoactive intestinal polypeptide (VIP)+ cells. In the dentate gyrus, a long-lasting deficit of somatostatin (SST)+ cells was observed. Both intranasal MSC and IGF1 therapy restored the majority of interneuron abnormalities in EoP mice. In line with the histological findings, EoP mice displayed impaired social behavior, which was partly restored by the therapies. In conclusion, induction of experimental EoP is associated with model-specific disturbances in interneuron development. In addition, intranasal MSCs and IGF1 are promising therapeutic strategies to aid interneuron development after EoP.

Origin and dynamics of oligodendrocytes in the developing brain: Implications for perinatal white matter injury.

Infants born prematurely are at high risk to develop white matter injury (WMI), due to exposure to hypoxic and/or inflammatory insults. Such perinatal insults negatively impact the maturation of oligodendrocytes (OLs), thereby causing deficits in myelination. To elucidate the precise pathophysiology underlying perinatal WMI, it is essential to fully understand the cellular mechanisms contributing to healthy/normal white matter development. OLs are responsible for myelination of axons. During brain development, OLs are generally derived from neuroepithelial zones, where neural stem cells committed to the OL lineage differentiate into OL precursor cells (OPCs). OPCs, in turn, develop into premyelinating OLs and finally mature into myelinating OLs. Recent studies revealed that OPCs develop in multiple waves and form potentially heterogeneous populations. Furthermore, it has been shown that myelination is a dynamic and plastic process with an excess of OPCs being generated and then abolished if not integrated into neural circuits. Myelination patterns between rodents and humans show high spatial and temporal similarity. Therefore, experimental studies on OL biology may provide novel insights into the pathophysiology of WMI in the preterm infant and offers new perspectives on potential treatments for these patients.

Combined fetal inflammation and postnatal hypoxia causes myelin deficits and autism-like behavior in a rat model of diffuse white matter injury.

Diffuse white matter injury (WMI) is a serious problem in extremely preterm infants, and is associated with adverse neurodevelopmental outcome, including cognitive impairments and an increased risk of autism-spectrum disorders. Important risk factors include fetal or perinatal inflammatory insults and fluctuating cerebral oxygenation. However, the exact mechanisms underlying diffuse WMI are not fully understood and no treatment options are currently available. The use of clinically relevant animal models is crucial to advance knowledge on the pathophysiology of diffuse WMI, allowing the definition of novel therapeutic targets. In the present study, we developed a multiple-hit animal model of diffuse WMI by combining fetal inflammation and postnatal hypoxia in rats. We characterized the effects on white matter development and functional outcome by immunohistochemistry, MRI and behavioral paradigms. Combined fetal inflammation and postnatal hypoxia resulted in delayed cortical myelination, microglia activation and astrogliosis at P18, together with long-term changes in oligodendrocyte maturation as observed in 10 week old animals. Furthermore, rats with WMI showed impaired motor performance, increased anxiety and signs of autism-like behavior, i.e. reduced social play behavior and increased repetitive grooming. In conclusion, the combination of fetal inflammation and postnatal hypoxia in rats induces a pattern of brain injury and functional impairments that closely resembles the clinical situation of diffuse WMI. This animal model provides the opportunity to elucidate pathophysiological mechanisms underlying WMI, and can be used to develop novel treatment options for diffuse WMI in preterm infants.

Promoting neuroregeneration after perinatal arterial ischemic stroke: neurotrophic factors and mesenchymal stem cells.

Newborns suffering from perinatal arterial ischemic stroke (PAIS) are at risk of neurodevelopmental problems. Current treatment options for PAIS are limited and mainly focus on supportive care, as presentation of PAIS is beyond the time window of current treatment strategies. Therefore, recent focus has shifted to interventions that stimulate regeneration of damaged brain tissue. From animal models, it is known that the brain increases its neurogenic capability after ischemic injury, by promoting neural cell proliferation and differentiation. However, neurogenesis is not maintained at the long term, which consequently impedes full repair leading to adverse consequences later in life. Boosting neuroregeneration of the newborn brain using treatment with neurotrophic factors and/or mesenchymal stem cells (MSCs) may be promising novel therapeutic strategies to improve neurological prospects and quality of life of infants with PAIS. This review focuses on effectiveness of neurotrophic growth factors, including erythropoietin, brain-derived neurotrophic factor, vascular endothelial growth factor, glial-derived neurotrophic factor, and MSC therapy, in both experimental neonatal stroke studies and first clinical trials for neonatal ischemic brain injury.

Delayed administration of neural stem cells after hypoxia-ischemia reduces sensorimotor deficits, cerebral lesion size, and neuroinflammation in neonatal mice.

BACKGROUND: Hypoxic-ischemic (HI) encephalopathy causes mortality and severe morbidity in neonates. Treatments with a therapeutic window >6 h are currently not available. Here, we explored whether delayed transplantation of allogenic neural stem cells (NSCs) at 10 d after HI could be a tool to repair HI brain injury and improve behavioral impairments. METHODS: HI was induced in 9-d-old mice. Animals received NSCs or vehicle intracranially in the hippocampus at 10 d post-HI. Sensorimotor performance was assessed by cylinder rearing test. Lesion size, synaptic integrity, and fate of injected NSCs were determined by immuno-stainings. Neuroinflammation was studied by immuno-stainings of brain sections, primary glial cultures, and TNFα ELISA. RESULTS: NSC transplantation at 10 d post-insult induced long-term improvement of motor performance and synaptic integrity, and reduced lesion size compared to vehicle-treatment. HI-induced neuroinflammation was reduced after NSC treatment, at least partially by factors secreted by NSCs. Injected NSCs migrated toward and localized at the damaged hippocampus. Transplanted NSCs differentiated toward the neuronal lineage and formed a niche with endogenous precursors. CONCLUSION: Our study provides evidence of the efficacy of NSC transplantation late after HI as a tool to reduce neonatal HI brain injury through regeneration of the lesion.

Repair of neonatal brain injury: bringing stem cell-based therapy into clinical practice.

Hypoxic-ischaemic brain injury is one of most important causes of neonatal mortality and long-term neurological morbidity in infants born at term. At present, only hypothermia in infants with perinatal hypoxic-ischaemic encephalopathy has shown benefit as a neuroprotective strategy. Otherwise, current treatment options for neonatal brain injury mainly focus on controlling (associated) symptoms. Regeneration of the injured neonatal brain with stem cell-based therapies is emerging and experimental results are promising. At present, increasing efforts are made to bring stem cell-based therapies to the clinic. Among all progenitor cell types, mesenchymal stromal (stem) cells seem to be most promising for human use given their neuroregenerative properties and favourable safety profile. This review summarizes the actual state, potential hurdles and possibilities of stem cell-based therapy for neonatal brain injury in the clinical setting. An early version of this paper was presented at the Groningen Early Intervention Meeting which was held in April 2016.

The neonatal brain is not protected by osteopontin peptide treatment after hypoxia-ischemia.

Neonatal encephalopathy due to perinatal hypoxia-ischemia (HI) is a severe condition, and current treatment options are limited. Expression of endogenous osteopontin (OPN), a multifunction glycoprotein, is strongly upregulated in the brain after neonatal HI. Intracerebrally administered OPN has been shown to be neuroprotective following experimental neonatal HI and adult stroke. In the present study, we determined whether intranasal, intraperitoneal or intracerebral treatment with a smaller TAT-OPN peptide is neuroprotective in neonatal mice with HI brain damage. The TAT-OPN peptide exerts bioactivity as it was as potent as full-length OPN in inducing cell adhesion in an in vitro adhesion assay. Intranasal administration of TAT-OPN peptide immediately after HI (T0) or in a repetitive treatment schedule of T0, 3 h, day (D) 1, 2 and 3 after HI did not protect cerebral gray or white matter after HI. Intraperitoneal TAT-OPN treatment at T0 or in two extended treatment schedules (D5, 7, 9, 11, 13, 15 after HI or T0, D1, 3, 5, 7, 9, 11, 13 and 15 after HI) did not result in neuroprotection either. Moreover, no functional improvement (cylinder rearing test and adhesive removal task) was observed following TAT-OPN treatment in any of the intraperitoneal treatment schedules. We validated that the TAT-OPN peptide reached the brain after intranasal or intraperitoneal administration by using an HIV-TAT staining. Finally, also intracerebral administration of the TAT-OPN peptide 1 h after HI did not reduce cerebral damage. Our data show that administration of the TAT-OPN peptide did not exert neuroprotective effects on neonatal HI-induced brain injury or sensorimotor behavioral deficits.

Development of cerebral gray and white matter injury and cerebral inflammation over time after inflammatory perinatal asphyxia.

Antenatal inflammation is associated with increased severity of hypoxic-ischemic (HI) encephalopathy and adverse outcome in human neonates and experimental rodents. We investigated the effect of lipopolysaccharide (LPS) on the timing of HI-induced cerebral tissue loss and gray matter injury, white matter injury and integrity, and the cerebral inflammatory response. On postnatal day 9, mice underwent HI by unilateral carotid artery occlusion followed by systemic hypoxia which resulted in early neuronal damage (MAP2 loss) at 3 h that did not increase up to day 15. LPS injection 14 h before HI (LPS+HI) significantly and gradually aggravated MAP2 loss from 3 h up to day 15, resulting in an acellular cystic lesion. LPS+HI increased white matter damage, reduced myelination in the corpus callosum and increased white matter fiber coherency in the cingulum. The number of oligodendrocytes throughout the lineage (Olig2-positive) was increased whereas more mature myelinating (CNPase-positive) oligodendrocytes were strongly decreased after LPS+HI. LPS+HI induced an increased and prolonged expression of cerebral cytokines/chemokines compared to HI. Additionally, LPS+HI increased macrophage/microglia activation and influx of neutrophils in the brain compared to HI. This study demonstrates the sensitizing effect of LPS on neonatal HI brain injury for an extended time-frame up to 15 days postinsult. LPS before HI induced a gradual increase in gray and white matter deficits, including reduced numbers of more mature myelinating oligodendrocytes and a decrease in white matter integrity. Moreover, LPS+HI prolonged and intensified the cerebral inflammatory response, including cellular infiltration. In conclusion, as the timing of damage and/or involved pathways are changed when HI is preceded by inflammation, experimental therapies might require modifications in the time window, dosage or combinations of therapies for efficacious neuroprotection.

Assessment of long-term safety and efficacy of intranasal mesenchymal stem cell treatment for neonatal brain injury in the mouse.

BACKGROUND: For clinical translation, we assessed whether intranasal mesenchymal stem cell (MSC) treatment after hypoxia-ischemia (HI) induces neoplasia in the brain or periphery at 14 mo. Furthermore, the long-term effects of MSCs on behavior and lesion size were determined. METHOD: HI was induced in 9-d-old mice. Pups received an intranasal administration of 0.5 × 10(6) MSCs or vehicle at 10 d post-HI. Full macroscopical and microscopical pathological analysis of 39 organs per mouse was performed. Sensorimotor behavior was assessed in the cylinder-rearing test at 10 d, 28 d, 6 mo, and 9 mo. Cognition was measured with the novel object recognition test at 3 and 14 mo post-HI. Lesion size was determined by analyzing mouse-anti-microtubule-associated protein 2 (MAP2) and mouse-anti-myelin basic protein (MBP) staining at 5 wk and 14 mo. RESULTS: At 14 mo post-HI, we did not observe any neoplasia in the nasal turbinates, brain, or other organs of HI mice treated with MSCs. Furthermore, our results show that MSC-induced improvement of sensorimotor and cognitive function is long lasting. In contrast, HI-vehicle mice showed severe behavioral impairment. Recovery of MAP2- and MBP-positive area lasted up to 14 mo following MSC treatment. CONCLUSION: Our results provide strong evidence of the long-term safety and positive effects of MSC treatment following neonatal HI in mice.

The rodent endovascular puncture model of subarachnoid hemorrhage: mechanisms of brain damage and therapeutic strategies.

Subarachnoid hemorrhage (SAH) represents a considerable health problem. To date, limited therapeutic options are available. In order to develop effective therapeutic strategies for SAH, the mechanisms involved in SAH brain damage should be fully explored. Here we review the mechanisms of SAH brain damage induced by the experimental endovascular puncture model. We have included a description of similarities and distinctions between experimental SAH in animals and human SAH pathology. Moreover, several novel treatment options to diminish SAH brain damage are discussed.SAH is accompanied by cerebral inflammation as demonstrated by an influx of inflammatory cells into the cerebral parenchyma, upregulation of inflammatory transcriptional pathways and increased expression of cytokines and chemokines. Additionally, various cell death pathways including cerebral apoptosis, necrosis, necroptosis and autophagy are involved in neuronal damage caused by SAH.Treatment strategies aiming at inhibition of inflammatory or cell death pathways demonstrate the importance of these mechanisms for survival after experimental SAH. Moreover, neuroregenerative therapies using stem cells are discussed as a possible strategy to repair the brain after SAH since this therapy may extend the window of treatment considerably. We propose the endovascular puncture model as a suitable animal model which resembles the human pathology of SAH and which could be applied to investigate novel therapeutic therapies to combat this debilitating insult.

G protein-coupled receptor kinase 2 and group I metabotropic glutamate receptors mediate inflammation-induced sensitization to excitotoxic neurodegeneration.

OBJECTIVE: The concept of inflammation-induced sensitization is emerging in the field of perinatal brain injury, stroke, Alzheimer disease, and multiple sclerosis. However, mechanisms underpinning this process remain unidentified. METHODS: We combined in vivo systemic lipopolysaccharide-induced or interleukin (IL)-1ß-induced sensitization of neonatal and adult rodent cortical neurons to excitotoxic neurodegeneration with in vitro IL-1ß sensitization of human and rodent neurons to excitotoxic neurodegeneration. Within these inflammation-induced sensitization models, we assessed metabotropic glutamate receptors (mGluR) signaling and regulation. RESULTS: We demonstrate for the first time that group I mGluRs mediate inflammation-induced sensitization to neuronal excitotoxicity in neonatal and adult neurons across species. Inflammation-induced G protein-coupled receptor kinase 2 (GRK2) downregulation and genetic deletion of GRK2 mimicked the sensitizing effect of inflammation on excitotoxic neurodegeneration. Thus, we identify GRK2 as a potential molecular link between inflammation and mGluR-mediated sensitization. INTERPRETATION: Collectively, our findings indicate that inflammation-induced sensitization is universal across species and ages and that group I mGluRs and GRK2 represent new avenues for neuroprotection in perinatal and adult neurological disorders.

Ultrasonic vocalization emission is altered following neonatal hypoxic-ischemic brain injury in mice.

Neonatal hypoxic-ischemic (HI) brain injury leads to cognitive impairments including social communication disabilities. Current treatments do not sufficiently target these impairments, therefore new tools are needed to examine social communication in models for neonatal brain injury. Ultrasonic vocalizations (USVs) during early life show potential as a measurement for social development and reflect landmark developmental stages in neonatal mice. However, changes in USV emission early after HI injury have not been found yet. Our current study examines USV patterns and classes in the first 3 days after HI injury. C57Bl/6 mice were subjected to HI on postnatal day (P)9 and USVs were recorded between P10 and P12. Audio files were analyzed using the VocalMat automated tool. HI-injured mice emitted less USVs, for shorter durations, and at a higher frequency compared to control (sham-operated) littermates. The HI-induced alterations in USVs were most distinct at P10 and in the frequency range of 50-75kHz. At P10 HI-injured mouse pups also produced different ratios of USV class types compared to control littermates. Moreover, alterations in the duration and frequency were specific to certain USV classes in HI animals compared to controls. Injury in the striatum and hippocampus contributed most to alterations in USV communication after HI. Overall, neonatal HI injury leads to USV alterations in newborn mice which could be used as a tool to study early HI-related social communication deficits.

CXCL10 is a crucial chemoattractant for efficient intranasal delivery of mesenchymal stem cells to the neonatal hypoxic-ischemic brain.

BACKGROUND: Hypoxic-Ischemic Encephalopathy (HIE) is a leading cause of mortality and morbidity in newborns. Recent research has shown promise in using intranasal mesenchymal stem cell (MSC) therapy if administered within 10 days after Hypoxia-Ischemia (HI) in neonatal mice. MSCs migrate from the nasal cavity to the cerebral lesion in response to chemotactic cues. Which exact chemokines are crucial for MSC guidance to the HI lesion is currently not fully understood. This study investigates the role of CXCL10 in MSC migration towards the HI-injured brain. METHODS: HI was induced in male and female 9-day-old C57BL/6 mice followed by intranasal MSC treatment at day 10 or 17 post-HI. CXCL10 protein levels, PKH26-labeled MSCs and lesion size were assessed by ELISA, immunofluorescent imaging and MAP2 staining respectively. At day 17 post-HI, when CXCL10 levels were reduced, intracranial CXCL10 injection and intranasal PKH26-labeled MSC administration were combined to assess CXCL10-guided MSC migration. MSC treatment efficacy was evaluated after 18 days, measuring lesion size, motor outcome (cylinder rearing task), glial scarring (GFAP staining) and neuronal density (NeuN staining) around the lesion. Expression of the receptor for CXCL10, i.e. CXCR3, on MSCs was confirmed by qPCR and Western Blot. Moreover, CXCL10-guided MSC migration was assessed through an in vitro transwell migration assay. RESULTS: Intranasal MSC treatment at day 17 post-HI did not reduce lesion size in contrast to earlier treatment timepoints. Cerebral CXCL10 levels were significantly decreased at 17 days versus 10 days post-HI and correlated with reduced MSC migration towards the brain. In vitro experiments demonstrated that CXCR3 receptor inhibition prevented CXCL10-guided migration of MSCs. Intracranial CXCL10 injection at day 17 post-HI significantly increased the number of MSCs reaching the lesion which was accompanied by repair of the HI lesion as measured by reduced lesion size and glial scarring, and an increased number of neurons around the lesion. CONCLUSIONS: This study underscores the crucial role of the chemoattractant CXCL10 in guiding MSCs to the HI lesion after intranasal administration. Strategies to enhance CXCR3-mediated migration of MSCs may improve the efficacy of MSC therapy or extend its regenerative therapeutic window.