TrkB-mediated neuroprotection in female hippocampal neurons is autonomous, estrogen receptor alpha-dependent, and eliminated by testosterone: a proposed model for sex differences in neonatal hippocampal neuronal injury.

BACKGROUND: Neonatal hypoxia ischemia (HI) related brain injury is one of the major causes of learning disabilities and memory deficits in children. In both human and animal studies, female neonate brains are less susceptible to HI than male brains. Phosphorylation of the nerve growth factor receptor TrkB has been shown to provide sex-specific neuroprotection following in vivo HI in female mice in an estrogen receptor alpha (ERα)-dependent manner. However, the molecular and cellular mechanisms conferring sex-specific neonatal neuroprotection remain incompletely understood. Here, we test whether female neonatal hippocampal neurons express autonomous neuroprotective properties and assess the ability of testosterone (T) to alter this phenotype. METHODS: We cultured sexed hippocampal neurons from ERα+/+ and ERα-/- mice and subjected them to 4 h oxygen glucose deprivation and 24 h reoxygenation (4-OGD/24-REOX). Sexed hippocampal neurons were treated either with vehicle control (VC) or the TrkB agonist 7,8-dihydroxyflavone (7,8-DHF) following in vitro ischemia. End points at 24 h REOX were TrkB phosphorylation (p-TrkB) and neuronal survival assessed by immunohistochemistry. In addition, in vitro ischemia-mediated ERα gene expression in hippocampal neurons were investigated following testosterone (T) pre-treatment and TrkB antagonist therapy via q-RTPCR. Multifactorial analysis of variance was conducted to test for significant differences between experimental conditions. RESULTS: Under normoxic conditions, administration of 3 µM 7,8-DHF resulted an ERα-dependent increase in p-TrkB immunoexpression that was higher in female, as compared to male neurons. Following 4-OGD/24-REOX, p-TrkB expression increased 20% in both male and female ERα+/+ neurons. However, with 3 µM 7,8-DHF treatment p-TrkB expression increased further in female neurons by 2.81 ± 0.79-fold and was ERα dependent. 4-OGD/24-REOX resulted in a 56% increase in cell death, but only female cells were rescued with 3 µM 7,8-DHF, again in an ERα dependent manner. Following 4-OGD/3-REOX, ERα mRNA increased ~ 3 fold in female neurons. This increase was blocked with either the TrkB antagonist ANA-12 or pre-treatment with T. Pre-treatment with T also blocked the 7,8-DHF- dependent sex-specific neuronal survival in female neurons following 4-OGD/24-REOX. CONCLUSIONS: OGD/REOX results in sex-dependent TrkB phosphorylation in female neurons that increases further with 7,8-DHF treatment. TrkB phosphorylation by 7,8-DHF increased ERα mRNA expression and promoted cell survival preferentially in female hippocampal neurons. The sex-dependent neuroprotective actions of 7,8-DHF were blocked by either ANA-12 or by T pre-treatment. These results are consistent with a model for a female-specific neuroprotective pathway in hippocampal neurons in response to hypoxia. The pathway is activated by 7,8-DHF, mediated by TrkB phosphorylation, dependent on ERα and blocked by pre-exposure to T.

Selectively compromised inner retina function following hypoxic-ischemic encephalopathy in mice: A noninvasive measure of severity of the injury.

The intricate system of connections between the eye and the brain implies that there are common pathways for the eye and brain that get activated following injury. Hypoxia-ischemia (HI) related encephalopathy is a consequence of brain injury caused by oxygen and blood flow deprivation that may result in visual disturbances and neurodevelopmental disorders in surviving neonates. We have previously shown that the tyrosine receptor kinase B (TrkB) agonist/modulator improves neuronal survival and long-term neuroprotection in a sexually differential way. In this study, we tested the hypotheses that; 1) TrkB agonist therapy improves the visual function in a sexually differential way; 2) Visual function detected by electroretinogram (ERG) correlates with severity of brain injury detected by magnetic resonance (MRI) imaging following neonatal HI in mice. To test our hypotheses, we used C57/BL6 mice at postnatal day (P) 9 and subjected them to either Vannucci's rodent model of neonatal HI or sham surgery. ERG was performed at P 30, 60, and 90. MRI was performed following the completion of the ERG. ERG in these mice showed that the a-wave is normal, but the b-wave amplitude is severely abnormal, reducing the b/a wave amplitude ratio. Inner retina function was found to be perturbed as we detected severely attenuated oscillatory potential after HI. No sex differences were detected in the injury and severity pattern to the retina as well as in response to 7,8-DHF therapy. Strong correlations were detected between the percent change in b/a ratio and percent hemispheric/hippocampal tissue loss obtained by MRI, suggesting that ERG is a valuable noninvasive tool that can predict the long-term severity of brain injury.

Retinal vascular recovery revealed by retinal imaging following neonatal hypoxia ischemia in mice: Is there a role for tyrosine kinase receptor modulation?

OBJECTIVE: Hypoxic ischemic encephalopathy (HIE) secondary to perinatal asphyxia leads to long-term visual disabilities. Dilated retinal exams in human newborns with HIE is an emerging diagnostic tool, but phenotypes of hypoxia ischemia (HI) related retinal vascular injury are unclear. 7,8-Dihydroxyflavone (7,8-DHF) is a TrkB agonist with protective effects on HI-related brain damage. We studied retinal vessels in a mouse model of neonatal HIE and the efficacy of 7,8-DHF in ameliorating HI-related retinal vascular injury. METHODS: C57BL6/J mice at post-natal day (P) 9 received unilateral left carotid artery ligation followed by exposure to 10 % oxygen for 50 min. Phosphate buffered saline or 7,8-DHF (5 mg/kg) were administered daily for 7 days intraperitoneally. Control groups of naïve or carotid artery ligation only mice were studied. Fluorescein angiography was performed in acute (two weeks post-exposure) and chronic (four weeks post-exposure) time points. Retinal artery width, retinal vein width, and collateral vessel length were quantified. RESULTS: Ligation of the common carotid artery alone caused retinal artery dilation in acute and chronic time points, but had no effect on retinal veins. At acute time point, HI caused increased retinal artery vasodilation, but was reversed by 7,8-DHF. HI caused short collateral vessel formation in ipsilateral eyes, rescued by 7,8-DHF treatment. CONCLUSION: Retinal artery vasodilation and collateral vessel formation due to HI were rescued by 7,8-DHF treatment. Retinal and collateral vessel monitoring could be diagnostic biomarkers for HI severity. Studies to elucidate mechanisms of 7,8-DHF action on retinal vessels could aid development of therapies for neonatal HI.

Sex differences in Hippocampal Memory and Learning following Neonatal Brain Injury: Is There a Role for Estrogen Receptor-α?

Neonatal encephalopathy due to hypoxia-ischemia (HI) leads to severe, life-long morbidities in thousands of neonates born in the USA and worldwide each year. Varying capacities of long-term episodic memory, verbal working memory, and learning can present without cerebral palsy and have been associated with the severity of neonatal encephalopathy sustained at birth. Among children who sustain a moderate degree of HI at birth, girls have larger hippocampal volumes compared to boys. Clinical studies indicate that female neonatal brains are more resistant to the effects of neonatal HI, resulting in better long-term cognitive outcomes compared to males with comparable brain injury. Our most recent mechanistic studies have addressed the origins and cellular basis of sex differences in hippocampal neuroprotection following neonatal HI-related brain injury and implicate estrogen receptor-α (ERα) in the neurotrophin receptor-mediated hippocampal neuroprotection in female mice. This review summarizes the recent findings on ERα-dependent, neurotrophin-mediated hippocampal neuroprotection and weighs the evidence that this mechanism plays an important role in preservation of long-term memory and learning following HI in females.

Developmental differences in microglia morphology and gene expression during normal brain development and in response to hypoxia-ischemia.

BACKGROUND: Neuroinflammation plays an important role in ischemic brain injury and recovery, however the interplay between brain development and the neuroinflammatory response is poorly understood. We previously described age-dependent differences in the microglial response and the effect of microglial inhibition. Here we investigate whether age-dependent microglial responses may be related to pre-injury developmental differences in microglial phenotype. METHODS: Measures of microglia morphology were quantified using semi-automated software analysis of immunostained sections from postnatal day 2 (P2), P9, P30 and P60 mice using IMARIS. Microglia were isolated from P2, P9, P30 and P60 mice, and expression of markers of classical and alternative microglial activation was assessed, as well as transforming growth factor beta (TGF-ß) receptor, Serpine1, Mer Tyrosine Kinase (MerTK), and the suppressor of cytokine signaling (SOCS3). Hypoxia-ischemia (HI) was induced in P9 and P30 mice using unilateral carotid artery ligation and exposure to 10% oxygen for 50 min. Microglia morphology and microglial expression of genes in the TGF-ß and MerTK pathways were determined in ipsilateral and contralateral hippocampus. RESULTS: A progressive and significant increase in microglia branching morphology was seen in all brain regions from P2 to P30. No consistent classical or alternative activation profile was seen in isolated microglia. A clear transition to increased expression of TGF-ß and its downstream effector serpine1 was seen between P9 and P30. A similar increase in expression was seen in MerTK and its downstream effector SOCS3. HI resulted in a significant decrease in branching morphology only in the P9 mice, and expression of TGF-ß receptor, Serpine1, MerTK, and SOCS3 were elevated in P30 mice compared to P9 post-HI. CONCLUSION: Microglia maturation is associated with changes in morphology and gene expression, and microglial responses to ischemia in the developing brain differ based on the age at which injury occurs.

Attenuation of Retinal Vascular Development in Neonatal Mice Subjected to Hypoxic-Ischemic Encephalopathy.

A significant proportion of children that survive hypoxic-ischemic encephalopathy (HIE) develop visual impairment. These visual deficits are generally attributed to injuries that occur in the primary visual cortex and other visual processing systems. Recent studies suggested that neuronal damage might also occur in the retina. An important structure affecting the viability of retinal neurons is the vasculature. However, the effects of HIE on the retinal neurovasculature have not been systemically evaluated. Here we investigated whether exposure of postnatal day 9 (P9) neonatal mice to HIE is sufficient to induce neurovascular damage in the retina. We demonstrate that the blood vessels on the surface of the retina, from mice subjected to HIE, were abnormally enlarged with signs of degeneration. The intermediate and deep vascular layers in these retinas failed to form normally, particularly in the periphery. All the vascular damages observed here were irreversible in nature up to 100 days post HIE. We also observed loss of retinal neurons, together with changes in both astrocytes and Müller cells mainly in the inner retina at the periphery. Collectively, our findings suggest that HIE results in profound alterations in the retinal vasculature, indicating the importance of developing therapeutic strategies to protect neurovascular dysfunction not only in the brain but also in the retina for infants exposed to HIE.

Identification of novel circulatory microRNA signatures linked to patients with ischemic stroke.

MicroRNAs (miRNAs) are involved in growth, development, and occurrence and progression of many diseases. MiRNA-mediated post-transcriptional regulation is poorly understood in vascular biology and pathology. The purpose of this is to determine circulatory miRNAs as early detectable peripheral biomarkers in patients with ischemic stroke (IS). MiRNAs expression levels were measured in IS serum samples and healthy controls using Illumina deep sequencing analysis and identified differentially expressed miRNAs. Differentially expressed miRNAs were further validated using SYBR-green-based quantitative real-time PCR (qRT-PCR) assay in postmortem IS brains, lymphoblastoid IS cell lines, oxygen and glucose deprivation/reoxygenation -treated human and mouse neuroblastoma cells, and mouse models of hypoxia and ischemia (HI)-induced stroke. A total of 4656 miRNAs were differentially expressed in IS serum samples relative to healthy controls. Out of 4656 miRNAs, 272 were found to be significantly deregulated in IS patients. Interestingly, we found several novel and previously unreported miRNAs in IS patients relative to healthy controls. Further analyses revealed that some candidate miRNAs and its target genes were involved in the regulation of the stroke. To the best of our knowledge, this is the first study identified potential novel candidate miRNAs in IS serum samples from the residents of rural West Texas. MiRNAs identified in this study could potentially be used as a biomarker and the development of novel therapeutic approaches for stroke. Further studies are necessary to better understand miRNAs-regulated stroke cellular changes.

Compatibility of MRI and FDG-PET findings with histopathological results in patients with focal cortical dysplasia.

PURPOSE: The present study aimed to determine if the specific characteristics of fluorodeoxyglucose-positron emission tomography (FDG-PET) analyses of the FCD subgroups were compatible with the magnetic resonance imaging (MRI) and clinical findings of the patients in these subgroups. METHODS: This study included 71 patients who had a presurgical evaluation workup performed due to drug-resistant seizures, who underwent epilepsy surgery, and who were histopathologically diagnosed with FCD. Relationships involving MRI and FDG-PET findings and clinical data from pathological subgroups and patients were assessed. RESULTS: According to the International League Against Epilepsy (ILAE) classifications of FCD, 28 of the patients were type I and 43 were type II. FCD was visible on the MRI scans of 53 patients, and a majority of this group was classified as type II FCD (n=34). Of these 53 patients, FCD was located in the temporal area of 21 patients, the extratemporal area of 29 patients. Of the patients who exhibited FDG-PET hypometabolism (PET-positive), 23 were classified as temporal, 17 as frontal, 11 showed involvement of the posterior cortex. The age of seizure onset was younger in PET-positive patients (p=0.032), and histopathological analyses revealed that 23 patients had type I FCD and 30 patients had type II FCD. CONCLUSION: PET scans reveal a lesion by showing hypometabolism in patients who have refractory epilepsy and an early age of onset with FCD. The lesions of MRI-negative/PET-positive FCD patients tend to be localized in the temporal lobe and that FCD may be localized in the frontal lobe of MRI-negative/PET-negative patients. However, the histopathological examinations of MRI-positive/PET-positive, MRI-negative/PET-positive, and MRI-negative/PET-negative patients did not exhibit a particular histopathological subtype.