Brain metabolism after therapeutic hypothermia for murine hypoxia-ischemia using hyperpolarized [1-13C] pyruvate magnetic resonance spectroscopy.

Hypoxic-ischemic encephalopathy (HIE) is a common neurological syndrome in newborns with high mortality and morbidity. Therapeutic hypothermia (TH), which is standard of care for HIE, mitigates brain injury by suppressing anaerobic metabolism. However, more than 40% of HIE neonates have a poor outcome, even after TH. This study aims to provide metabolic biomarkers for predicting the outcomes of hypoxia-ischemia (HI) after TH using hyperpolarized [1-13C] pyruvate magnetic resonance spectroscopy. Postnatal day 10 (P10) mice with HI underwent TH at 1 h and were scanned at 6-8 h (P10), 24 h (P11), 7 days (P17), and 21 days (P31) post-HI on a 14.1-T NMR spectrometer. The metabolic images were collected, and the conversion rate from pyruvate to lactate and the ratio of lactate to pyruvate in the injured left hemisphere (kPL(L) and Lac/Pyr(L), respectively) were calculated at each timepoint. The outcomes of TH were determined by the assessments of brain injury on T2-weighted images and behavioral tests at later timepoint. kPL(L) and Lac/Pyr(L) over time between the good-outcome and poor-outcome groups and across timepoints within groups were analyzed. We found significant differences in temporal trends of kPL(L) and Lac/Pyr(L) between groups. In the good-outcome group, kPL(L) increased until P31 with a significantly higher value at P31 compared with that at P10, while the level of Lac/Pyr(L) at P31 was notably higher than those at all other timepoints. In the poor-outcome group, kPL(L) and Lac/Pyr(L) increased within 24 h. The kPL(L) value at P11 was considerably higher compared with P10. Discrete temporal changes of kPL(L) and Lac/Pyr(L) after TH between the good-outcome and poor-outcome groups were seen as early as 24 h after HI, reflecting various TH effects on brain anaerobic metabolism, which may provide insights for early screening for response to TH.

Reduced folate carrier 1 is present in retinal microvessels and crucial for the inner blood retinal barrier integrity.

BACKGROUND: Reduced folate carrier 1 (RFC1; SLC19a1) is the main responsible transporter for the B9 family of vitamins named folates, which are essential for normal tissue growth and development. While folate deficiency resulted in retinal vasculopathy, the expression and the role of RFC1 in blood-retinal barrier (BRB) are not well known. METHODS: We used whole mount retinas and trypsin digested microvessel samples of adult mice. To knockdown RFC1, we delivered RFC1-targeted short interfering RNA (RFC1-siRNA) intravitreally; while, to upregulate RFC1 we delivered lentiviral vector overexpressing RFC1. Retinal ischemia was induced 1-h by applying FeCl3 to central retinal artery. We used RT-qPCR and Western blotting to determine RFC1. Endothelium (CD31), pericytes (PDGFR-beta, CD13, NG2), tight-junctions (Occludin, Claudin-5 and ZO-1), main basal membrane protein (Collagen-4), endogenous IgG and RFC1 were determined immunohistochemically. RESULTS: Our analyses on whole mount retinas and trypsin digested microvessel samples of adult mice revealed the presence of RFC1 in the inner BRB and colocalization with endothelial cells and pericytes. Knocking down RFC1 expression via siRNA delivery resulted in the disintegration of tight junction proteins and collagen-4 in twenty-four hours, which was accompanied by significant endogenous IgG extravasation. This indicated the impairment of BRB integrity after an abrupt RFC1 decrease. Furthermore, lentiviral vector-mediated RFC1 overexpression resulted in increased tight junction proteins and collagen-4, confirming the structural role of RFC1 in the inner BRB. Acute retinal ischemia decreased collagen-4 and occludin levels and led to an increase in RFC1. Besides, the pre-ischemic overexpression of RFC1 partially rescued collagen-4 and occludin levels which would be decreased after ischemia. CONCLUSION: In conclusion, our study clarifies the presence of RFC1 protein in the inner BRB, which has recently been defined as hypoxia-immune-related gene in other tissues and offers a novel perspective of retinal RFC1. Hence, other than being a folate carrier, RFC1 is an acute regulator of the inner BRB in healthy and ischemic retinas.

Importance of Pericytes in the Pathophysiology of Cerebral Ischemia.

Various cell types contribute to pathological changes observed in the brain following cerebral ischemia. Pericytes, as a component of neurovascular unit (NVU) and blood brain barrier (BBB), play a key role for cerebral blood flow control and regulation of vessel permeability. It was shown that pericytes can control cerebral blood flow at the level of capillaries, by their contractile property. Their role in BBB development and maintenance are crucial for guidance of brain vessel development, new vessel formation and stabilization of the newly formed vessels. Additionally, they can contribute to inflammation in response to inflammatory stimuli and can differentiate to various cell types by their multipotent differentiation properties. This cell type which is intimately associated with cerebral circulation also plays important roles during cerebral ischemia. Here, we review the properties and physiological functions of pericytes, how these functions change during ischemia to affect the pathophysiology of ischemic stroke and post stroke cognitive impairment. Pericytes are a neglected cell type and they are not unambiguously characterized which in turn led to contradictory findings in the literature. Clear characterization of pericytes by current methods will help better understanding of their role in the pathophysiology of stroke. With the information gained from these efforts it will be possible to develop pericyte specific therapeutic targets and achieve important breakthroughs in clinical recovery in ischemic stroke treatment.

Clinical evaluation of muscle functions in neurofibromatosis type 1.

AIM: Muscle weakness, fatigue and speech problems can occur in neurofibromatosis type 1 (NF1). The pathogenesis of these symptoms is unclear, likely multifactorial. We examined motor function in limb and speech muscles in NF1 patients. METHODS: We evaluated NF1 and control groups aged 4-18 years for muscle strength, tone and mobility using standard manual testing, joint motion and Beighton score measurements. Speech and language functions were assessed by speech articulation and resonance. As a marker of muscle tissue turnover, we determined collagen degradation products in urine before and after submaximal exercise. RESULTS: NF1 patients had reduced strength in proximal limb muscles compared to control subjects. Speech articulation problems and hypernasality were more common in NF1 (47% and 38%, respectively). Collagen products excreted in urine correlated with gluteal and biceps muscle strength. CONCLUSION: Muscle dysfunction can be detected in some children with NF1 and may explain certain clinical features including fatigue, speech and articulation problems. If confirmed by further research, these findings may be relevant to the management of this condition.

Epilepsy in neurofibromatosis type 1: Diffuse cerebral dysfunction?

INTRODUCTION: Neurofibromatosis type 1 (NF1) is accompanied by epileptic seizures in 4-7% of patients. We examined clinical, electrophysiological, and radiological features associated with epilepsy in our NF1 series in order to identify risk factors. METHODS: We reviewed data of 641 pediatric patients with NF1 diagnosis according to National Institutes of Health (NIH) criteria in Hacettepe University records from January 2008-August 2018. Demographic features, NF1-related clinical and imaging characteristics, age at onset of epilepsy, seizure semiology, and frequency, electroencephalogram (EEG) findings, and response to treatment were noted. RESULTS: Twenty-six patients with NF1, 15 male, 11 female, had epilepsy. Age at seizure onset was 6 months to 13 years. Seizure semiology was focal with impaired awareness (n = 9, 34%), focal aware motor (n = 2, 8%), focal to bilateral tonic-clonic (n = 3, 12%), generalized tonic-clonic (n = 7, 28%), absence (n = 3, 12%), infantile spasms (n = 1), and unclassified type (n = 1). None had a history of status epilepticus. The EEG findings were normal for age in ten patients (38%). Others had focal (n = 8, 30%), generalized (n = 7, 27%), or multifocal (n = 1, 4%) discharges. On brain magnetic resonance imaging (MRI) signal intensity changes typical for NF1 (neurofibromatosis bright objects, NBOs) were the most common finding (80%), followed by normal MRI (20%). There was no relation between the localization of NBOs and discharges on EEG. Seventeen patients (65%) were seizure-free at the time of the study; 11 of them still under medication including four on multiple antiepileptic drugs. The rate of learning problems and NBO were significantly higher in patients with NF1 with epilepsy compared to those without. DISCUSSION: Epilepsy in NF1 is associated with relatively infrequent seizures and good response to treatment. Learning disorders are markedly frequent in this group, irrespective of the severity of epilepsy. The absence of correlation between the localizations of epileptiform discharges and lesions on MRI support the role of cellular or synaptic mechanisms rather than structural causes in the pathogenesis of epilepsy.