Neuroinvasion and anosmia are independent phenomena upon infection with SARS-CoV-2 and its variants.

Anosmia was identified as a hallmark of COVID-19 early in the pandemic, however, with the emergence of variants of concern, the clinical profile induced by SARS-CoV-2 infection has changed, with anosmia being less frequent. Here, we assessed the clinical, olfactory and neuroinflammatory conditions of golden hamsters infected with the original Wuhan SARS-CoV-2 strain, its isogenic ORF7-deletion mutant and three variants: Gamma, Delta, and Omicron/BA.1. We show that infected animals develop a variant-dependent clinical disease including anosmia, and that the ORF7 of SARS-CoV-2 contributes to the induction of olfactory dysfunction. Conversely, all SARS-CoV-2 variants are neuroinvasive, regardless of the clinical presentation they induce. Taken together, this confirms that neuroinvasion and anosmia are independent phenomena upon SARS-CoV-2 infection. Using newly generated nanoluciferase-expressing SARS-CoV-2, we validate the olfactory pathway as a major entry point into the brain in vivo and demonstrate in vitro that SARS-CoV-2 travels retrogradely and anterogradely along axons in microfluidic neuron-epithelial networks.

Bacterial sensing via neuronal Nod2 regulates appetite and body temperature.

Gut bacteria influence brain functions and metabolism. We investigated whether this influence can be mediated by direct sensing of bacterial cell wall components by brain neurons. In mice, we found that bacterial peptidoglycan plays a major role in mediating gut-brain communication via the Nod2 receptor. Peptidoglycan-derived muropeptides reach the brain and alter the activity of a subset of brain neurons that express Nod2. Activation of Nod2 in hypothalamic inhibitory neurons is essential for proper appetite and body temperature control, primarily in females. This study identifies a microbe-sensing mechanism that regulates feeding behavior and host metabolism.

Tissue clearing and 3D imaging in developmental biology.

Tissue clearing increases the transparency of late developmental stages and enables deep imaging in fixed organisms. Successful implementation of these methodologies requires a good grasp of sample processing, imaging and the possibilities offered by image analysis. In this Primer, we highlight how tissue clearing can revolutionize the histological analysis of developmental processes and we advise on how to implement effective clearing protocols, imaging strategies and analysis methods for developmental biology.

Mapping the Fine-Scale Organization and Plasticity of the Brain Vasculature.

The cerebral vasculature is a dense network of arteries, capillaries, and veins. Quantifying variations of the vascular organization across individuals, brain regions, or disease models is challenging. We used immunolabeling and tissue clearing to image the vascular network of adult mouse brains and developed a pipeline to segment terabyte-sized multichannel images from light sheet microscopy, enabling the construction, analysis, and visualization of vascular graphs composed of over 100 million vessel segments. We generated datasets from over 20 mouse brains, with labeled arteries, veins, and capillaries according to their anatomical regions. We characterized the organization of the vascular network across brain regions, highlighting local adaptations and functional correlates. We propose a classification of cortical regions based on the vascular topology. Finally, we analysed brain-wide rearrangements of the vasculature in animal models of congenital deafness and ischemic stroke, revealing that vascular plasticity and remodeling adopt diverging rules in different models.

Cold stress protein RBM3 responds to hypothermia and is associated with good stroke outcome.

RNA-binding motif protein 3 is a molecular marker of hypothermia that has proved neuroprotective in neurodegenerative disease models. However, its relationship to the well-recognized therapeutic effect of hypothermia in ischaemic stroke had not been studied. In this work, the expression of RNA-binding motif protein 3 was investigated in ischaemic animal models subjected to systemic and focal brain hypothermia, specifically the effects of RNA-binding motif protein 3 silencing and overexpression on ischaemic lesions. Moreover, the association of RNA-binding motif protein 3 levels with body temperature and clinical outcome was evaluated in two independent cohorts of acute ischaemic stroke patients (n = 215); these levels were also determined in a third cohort of 31 patients derived from the phase III EuroHYP-1 trial of therapeutic cooling in ischaemic stroke. The preclinical data confirmed the increase of brain RNA-binding motif protein 3 levels in ischaemic animals subjected to systemic and focal hypothermia; this increase was selectively higher in the cooled hemisphere of animals undergoing focal brain hypothermia, thus confirming the direct effect of hypothermia on RNA-binding motif protein 3 expression, while RNA-binding motif protein 3 up-regulation in ischaemic brain regions led to functional recovery. Clinically, patients with body temperature <37.5°C in the first two cohorts had higher RNA-binding motif protein 3 values at 24 h and good outcome at 3 months post-ischaemic stroke, while RNA-binding motif protein 3 levels in the cooled third cohort tended to exceed those in placebo-treated patients. These results make RNA-binding motif protein 3 a molecular marker associated with the effect of hypothermia in ischaemic stroke and suggest its potential application as a promising protective target.

Light-Emitting Diode Photobiomodulation After Cerebral Ischemia.

Photobiomodulation (PBM) therapy is a promising therapeutic approach for several pathologies, including stroke. The biological effects of PBM for the treatment of cerebral ischemia have previously been explored as a neuroprotective strategy using different light sources, wavelengths, and incident light powers. However, the capability of PBM as a novel alternative therapy to stimulate the recovery of the injured neuronal tissue after ischemic stroke has been poorly explored. The aim of this study was to investigate the low-level light irradiation therapy by using Light Emitting Diodes (LEDs) as potential therapeutic strategy for stroke. The LED photobiomodulation (continuous wave, 830 nm, 0.2-0.6 J/cm2) was firstly evaluated at different energy densities in C17.2 immortalized mouse neural progenitor cell lines, in order to observe if this treatment had any effect on cells, in terms of proliferation and viability. Then, the PBM-LED effect (continuous wave, 830 nm, 0.28 J/cm2 at brain cortex) on long-term recovery (12 weeks) was analyzed in ischemic animal model by means lesion reduction, behavioral deficits, and functional magnetic resonance imaging (fMRI). Analysis of cellular proliferation after PBM was significantly increased (1 mW) in all different exposure times used; however, this effect could not be replicated in vivo experimental conditions, as PBM did not show an infarct reduction or functional recovery. Despite the promising therapeutic effect described for PBM, further preclinical studies are necessary to optimize the therapeutic window of this novel therapy, in terms of the mechanism associated to neurorecovery and to reduce the risk of failure in futures clinical trials.

Iron deposition in periaqueductal gray matter as a potential biomarker for chronic migraine.

OBJECTIVE: To study iron deposition in red nucleus (RN), globus pallidus (GP), and periaqueductal gray matter (PAG) as a potential biomarker of chronic migraine (CM) and its association with levels of biomarkers related to migraine pathophysiology. METHODS: This case-control study included 112 patients with migraine (55 CM, 57 episodic migraine [EM]) and 25 headache-free controls. We analyzed iron deposition using 3T MRI and the NIH software platform ImageJ; we analyzed serum levels of markers of inflammation, endothelial dysfunction, and blood-brain barrier (BBB) disruption by ELISA in peripheral blood during interictal periods. RESULTS: Patients with CM showed larger iron grounds volume in RN compared to patients with EM (70.2 ± 6.8 vs 25.5 ± 7.3 µL, p < 0.001) and controls (70.2 ± 6.8 vs 15.1 ± 10.8 µL, p < 0.001), as well as larger iron deposits in PAG compared to patients with EM (360.3 ± 6.5 vs 249.7 ± 6.9 µL, p < 0.001) and controls (360.3 ± 6.5 vs 168.6 ± 10.3 µL, p < 0.001). In PAG, differences were also significant between patients with EM and controls. No significant differences were obtained for GP. Receiver operating characteristic curves showed that the optimal threshold for iron volume was 15 µL in RN (80% sensitivity, 71% specificity) and 240 µL in PAG (93% sensitivity, 97% specificity). Iron grounds volume in PAG was correlated with higher plasma levels of soluble tumor necrosis factor-like WEAK (r = 0.395, p = 0.005) and cellular fibronectin (r = 0.294, p = 0.040). CONCLUSIONS: Patients with CM showed increased iron deposition in RN and PAG compared to patients with EM and controls. Iron grounds volume in PAG identified correctly patients with CM and was associated with elevated biomarkers of endothelial dysfunction and BBB disruption.

Blood glutamate EAAT2-cell grabbing therapy in cerebral ischemia.

BACKGROUND: Excitatory amino acid transporter 2 (EAAT2) plays a pivotal role in glutamate clearance in the adult brain, thereby preventing excitotoxic effects. Considering the high efficacy of EAAT2 for glutamate uptake, we hypothesized that the expression of this transporter in mesenchymal stem cells (MSCs) for systemic administration could yield a cell-based glutamate-grabbing therapy, combining the intrinsic properties of these cells with excitotoxic protection. METHODS: To address this hypothesis, EAAT2-encoding cDNA was introduced into MSCs and human embryonic kidney 293 cells (HEK cells) as the control cell line. EAAT2 expression and functionality were evaluated by in vitro assays. Blood glutamate-grabbing activity was tested in healthy and ischemic rat models treated with 3 × 106 and 9 × 106 cells/animal. FINDINGS: The expression of EAAT2 in both cell types conferred the expected glutamate-grabbing activity in in vitro and in vivo studies. The functional improvement observed in ischemic rats treated with EAAT2-HEK at low dose, confirmed that this effect was indeed mediated by the glutamate-grabbing activity associated with EAAT2 functionality. Unexpectedly, both cell doses of non-transfected MSCs induced higher protection than transfected EAAT2-MSCs by another mechanism independent of the glutamate-grabbing capacity. INTERPRETATION: Although the transfection procedure most likely interferes with some of the intrinsic protective mechanisms of mesenchymal cells, the results show that the induced expression of EAAT2 in cells represents a novel alternative to mitigate the excitotoxic effects of glutamate and paves the way to combine this strategy with current cell therapies for cerebral ischemia.

CGRP and PTX3 as Predictors of Efficacy of Onabotulinumtoxin Type A in Chronic Migraine: An Observational Study.

OBJECTIVE: The aim of this study is to find a relation between several biomarkers in peripheral blood and outcome after treatment with onabotulinumtoxin A (OnabotA). BACKGROUND: OnabotA is an effective treatment in chronic migraine (CM). Different studies have tried to find predictors of response to treatment, either with clinical characteristics, neuroimaging features, or molecular biomarkers; however, it is still not possible to predict the individual outcome. METHODS: We measured serum levels of biomarkers of inflammation (IL-6, IL-10, TNF-α, and hs-CRP), endothelial dysfunction (PTX3 and sTWEAK), blood-brain barrier disruption (cFN), brain damage (S100b, NSE), and trigemino-vascular activation (CGRP) by ELISA in a group of CM patients treated with OnabotA and healthy controls. After 24 weeks, patients were classified in two groups according to their outcome considering variations in headache frequency: nonresponders (nonimprovement or improvement <50%) and responders (improvement >50%). We compared baseline levels of biomarkers between these groups. RESULTS: Sixty-two patients diagnosed with CM (IHS 2013 criteria) who fulfilled criteria for treatment with OnabotA and 24 healthy controls were included. Fifteen patients did not respond to treatment (24.2%) and 47 were responders (75.8%). Pentraxin 3 (PTX3) serum levels (1455.4 ± 487.5 pg/mL versus 720.3 ± 334.1 pg/mL, P < .0001) and calcitonin gene-related peptide (CGRP) serum levels (133.1 ± 86.6 ng/mL versus 58.2 ± 91.7 ng/mL, P = .004) were significantly higher in responders than nonresponders. Serum basal levels of PTX3 >1000 pg/mL (AUC 0.908; 95% CI: 0.827-0.990) and CGRP >50 ng/mL (AUC 0.800; 95% CI: 0.652-0.947) were associated with good response to OnabotA treatment. CONCLUSIONS: These results show that molecular markers of trigeminovascular activation (CGRP) and endothelial dysfunction (PTX3) are associated with response to OnabotA and may act as new biomarkers for the selection of treatment in chronic migraineurs.

Role of adipocytokines in the pathophysiology of migraine: A cross-sectional study.

Background Obesity is a risk factor for migraine and headache chronification. Adipocytokines may be involved in this correlation. Objective To relate serum adipocytokine levels to clinical and biochemical parameters associated with migraine. Methods We measured levels of leptin, adiponectin and other inflammatory (interleukin 6, interleukin 10, tumor necrosis factor α, high sensitivity C-reactive protein) and endothelial (pentraxin 3, soluble TNF-like weak inducer of apoptosis) molecules potentially related to migraine pathophysiology in a group of migraine patients (IHS 2013) and healthy controls. Results One hundred and eleven patients (mean age 39.7 years, 93% female) and 24 healthy controls (mean age 35.9 years, 90% female) were included. Fifty-six patients were diagnosed with episodic migraine (mean age 35.1 years, 98.2% female) and 55 patients with chronic migraine (mean age 44.4 years, 89.5% female). Leptin serum levels (15.2 ng/mL, SD = 10.5 vs . 3.1 ng/mL, SD = 0.9; p < 0.001) and adiponectin serum levels (72.3 µg/mL, SD = 38.5 vs . 37.7 µg/mL, SD = 16.9; p < 0.001) were significantly increased in migraine patients. Leptin serum levels (15.5 ng/mL, SD = 9.7 vs . 10.8 ng/mL, SD = 6.0; p < 0.001) and adiponectin serum levels (65.8 µg/mL, SD = 42.9 vs . 33.2 µg/mL, SD = 31.0; p < 0.001) were significantly higher in chronic compared to episodic migraine patients. We found a positive correlation between leptin levels and inflammatory biomarkers: IL6 (r = 0.498; p < 0.001), TNF-α (r = 0.389; p < 0.001), and hs-CRP (r = 0.422; p < 0.001). Conclusions Leptin and adiponectin are increased in migraineurs. There is a correlation between adipocytokine levels and other inflammation-related molecules. This suggests a potential role of adipocytokines in migraine pathophysiology and chronification.

Higher Expression of Toll-Like Receptors 2 and 4 in Blood Cells of Keratoconus Patiens.

Inflammation may play a significant role in Keratoconus (KC), but the implication of immunity on this inflammatory response is unknown. Therefore, our aim was to determine the expression levels of Toll-like receptors 2 (TLR2) and 4 (TLR4) in monocytes and neutrophils from patients with KC and control subjects for demonstrating the role of innate immunity in KC. We also study the correlation between TLR2/TLR4 expression and serum levels of proinflammatory markers (IL-1ß, IL-6, TNF-α, MMP-9 and NF-κB). Forty patients with bilateral KC (55% males; mean age; 33.1 ± 10.9 years) and 20 control subjects (55% males; mean age; 30.4 ± 7.6 years) were included in the study. Our results showed that mean expression of TLR2 and TLR4 in both neutrophils and monocytes was significantly higher in patients with KC compared to control subjects (all p < 0.0001). Furthermore, KC patients also showed higher serum levels of IL-1B, IL-6, TNF-α, MMP-9 (all p < 0.0001) and NF-κB (p = 0.036). In addition, we found a strong correlation between TLR2 expression in both monocytes and neutrophils (all p < 0.0001), and TLR4 in monocytes (all p < 0.05) with serum levels of IL-1B, IL-6, TNF-α and MMP-9. In conclusion, these findings suggest that TLRs may play an important role in the pathophysiology of KC.

CM352 Reduces Brain Damage and Improves Functional Recovery in a Rat Model of Intracerebral Hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) is an acute neurological disorder with high mortality and no effective treatment. In addition to the initial bleeding event, rebleeding and hematoma expansion are associated with poor outcome in these patients. We studied the effectiveness of the new antifibrinolytic agent CM352, a short-half-life matrix metalloproteinase inhibitor, for achieving early hemostasis and improving functional recovery in a rat model of collagenase-induced ICH. METHODS AND RESULTS: ICH was induced by striatal injection of collagenase, and 1 hour later, rats received an intravenous injection of saline (n=6) or CM352 (1 mg/kg, n=6). Hematoma (basal and after 3 and 24 hours) and lesion (14 days) volumes were quantified on T2-weighted (T2) magnetic resonance images. Neurological and functional recovery was evaluated by using Bederson score and a cylinder test (basal, 24 hours, and 14 days). Early treatment (1 hour) with CM352 was efficient reducing hematoma expansion at 3 hours (P<0.01) and, more markedly, at 24 hours (P<0.01). Decreased bleeding after antifibrinolytic treatment was accompanied by reduced interleukin-6 levels at 3 hours (P<0.05) and smaller lesion volume at 14 days (P<0.01). CM352 drastically reduced sensorimotor impairment (cylinder test) after ICH in rats at 24 hours (P<0.01) and 14 days (P<0.01). Similarly, it also attenuated neurological deficit (Bederson scale) at 24 hours (P<0.01) and 14 days (P<0.01). Interestingly, late (3 hours) CM352 administration also resulted in reduced lesion size and better functional outcome. CONCLUSIONS: CM352, a new antifibrinolytic agent and matrix metalloproteinase inhibitor, effectively prevented hematoma growth and reduced lesion size in ICH in association with improved functional and neurological recovery.

Preclinical PET Study of Intravitreal Injections.

Purpose: This work aimed at describing the time course of vitreous clearance through the use of positron emission tomography (PET) as a noninvasive tool for pharmacokinetic studies of intravitreal injection. Methods: The pharmacokinetic profile of intravitreal injections of molecules labeled with 18Fluorine (18F) was evaluated in adult Sprague Dawley rats by using a dedicated small-animal PET/computed tomography scanner. Different conditions were studied: three molecules radiolabeled with 18F (18F-FDG, 18F-NaF, and 18F-Choline), three volumes of intravitreal injections (7, 4, and 2 µL), and absence or presence of eye inflammation (uveitis). Results: Our results showed that there are significant pharmacokinetic differences among the radiolabeled molecules studied but not among the injected volumes. The presence or absence of uveitis was an important factor in vitreous clearance, since the elimination of the drug was clearly increased when this condition is present. Conclusions: Intravitreal pharmacokinetic studies based on the use of dedicated PET imaging can be of potential interest as noninvasive tools in ophthalmic drug development in small animals.

Vectorized nanodelivery systems for ischemic stroke: a concept and a need.

Neurological diseases of diverse aetiologies have significant effects on the quality of life of patients. The limited self-repairing capacity of the brain is considered to be the origin of the irreversible and progressive nature of many neurological diseases. Therefore, neuroprotection is an important goal shared by many clinical neurologists and neuroscientists. In this review, we discuss the main obstacles that have prevented the implementation of experimental neuroprotective strategies in humans and propose alternative avenues for the use of neuroprotection as a feasible therapeutic approach. Special attention is devoted to nanotechnology, which is a new approach for developing highly specific and localized biomedical solutions for the study of the multiple mechanisms involved in stroke. Nanotechnology is contributing to personalized neuroprotection by allowing us to identify mechanisms, determine optimal therapeutic windows, and protect patients from brain damage. In summary, multiple aspects of these new players in biomedicine should be considered in future in vivo and in vitro studies with the aim of improving their applicability to clinical studies.

Positron Emission Tomography for the Development and Characterization of Corneal Permanence of Ophthalmic Pharmaceutical Formulations.

Purpose: This work is aimed at describing the utility of positron emission tomography/computed tomography (PET/CT) as a noninvasive tool for pharmacokinetic studies of biopermanence of topical ocular formulations. Methods: The corneal biopermanence of a topical ophthalmic formulation containing gellan gum and kappa carragenan (0.82% wt/vol) labeled with 18Fluorine (18F) radiotracers (18F-FDG and 18F-NaF) was evaluated by using a dedicated small-animal PET/CT, and compared with the biopermanence of an aqueous solution labeled with the same compounds. Regions of interest (ROIs) were manually drawn on the reconstructed PET images for quantifying the radioactivity concentration in the eye. The biopermanence of the formulations was determined by measuring the radioactivity concentration at different times after topical application. Additionally, cellular and ex vivo safety assays were performed to assess the safety of the performed procedures. Results: Differences were observed in the ocular pharmacokinetics of the two formulations. After 1.5 hours of contact, 90% of the hydrogel remained in the ocular surface, while only 69% of the control solution remained. Furthermore, it was observed that flickering had a very important role in the approach of the trial. The application of 18F-FDG in the eye was neither irritating nor cytotoxic for human corneal epithelial cells. Conclusions: The use of small-animal PET and 18F radiotracers in ocular pharmacokinetics of ophthalmic formulations is feasible and could be a safe method for future ocular pharmacokinetic studies in humans.

In vivo eye surface residence determination by high-resolution scintigraphy of a novel ion-sensitive hydrogel based on gellan gum and kappa-carrageenan.

In last years, sensitive hydrogels have become a breakthrough in ophthalmic pharmaceutical technology aimed at developing new strategies to increase the residence time of active substances. In a previous paper, we qualitatively demonstrated the capacity of a new ion sensitive hydrogel to increase the residence time. Nevertheless, the clearance of the gel from the ocular surface was not quantifiable with the used methodology. The aim of the present work was to use a well-established approach based on scintigraphy to quantitatively estimate the residence time of the previously proposed hydrogel. The rat corneal residence time of a topic ophthalmic formulation containing gellan gum and kappa carragenan (0.82% w/v) labeled with 99mTc-DTPA radiotracer was evaluated and compared with the residence of an aqueous solution. Ophthalmic safety studies such as eye irritation or passage through the cornea were also carried out. After 1.5h of contact, 77% of the hydrogel remained in the ocular surface, presenting kinetics of disappearance one-phase decay and a half time of 262min. We conclude that the novel ophthalmic hydrogel developed with kappa carrageenan and gellan gum remains for long periods of time on the corneal surface, presenting a drop that fits an exponential decay.

Intraarterial route increases the risk of cerebral lesions after mesenchymal cell administration in animal model of ischemia.

Mesenchymal stem cells (MSCs) are a promising clinical therapy for ischemic stroke. However, critical parameters, such as the most effective administration route, remain unclear. Intravenous (i.v.) and intraarterial (i.a.) delivery routes have yielded varied outcomes across studies, potentially due to the unknown MSCs distribution. We investigated whether MSCs reached the brain following i.a. or i.v. administration after transient cerebral ischemia in rats, and evaluated the therapeutic effects of both routes. MSCs were labeled with dextran-coated superparamagnetic nanoparticles for magnetic resonance imaging (MRI) cell tracking, transmission electron microscopy and immunohistological analysis. MSCs were found in the brain following i.a. but not i.v. administration. However, the i.a. route increased the risk of cerebral lesions and did not improve functional recovery. The i.v. delivery is safe but MCS do not reach the brain tissue, implying that treatment benefits observed for this route are not attributable to brain MCS engrafting after stroke.

Protective Effects and Magnetic Resonance Imaging Temperature Mapping of Systemic and Focal Hypothermia in Cerebral Ischemia.

BACKGROUND AND PURPOSE: Hypothermia is potentially the most effective protective therapy for brain ischemia; however, its use is limited because of serious side effects. Although focal hypothermia (FH) has a significantly lower stress profile than systemic hypothermia (SH), its efficacy in ischemia has been poorly studied. We aimed to compare the therapeutic effects of each treatment on various short- and long-term clinically relevant end points. METHODS: Sprague-Dawley rats were subjected to transient (45 minutes) occlusion of the middle cerebral artery. One hour after arterial reperfusion, animals were randomly assigned to groups for treatment with SH or FH (target temperature: 32°C) for 4 or 24 hours. Lesion volume, edema, functional recovery, and histological markers of cellular injury were evaluated for 1 month after ischemic injury. Effects of SH and FH on cerebral temperature were also analyzed for the first time by magnetic resonance thermometry, an approach that combines spectroscopy with gradient-echo-based phase mapping. RESULTS: Both therapeutic approaches reduced ischemic lesion volume (P<0.001), although a longer FH treatment (24 hours) was required to achieve similar protective effects to those induced by 4 hours of SH. In addition, magnetic resonance thermometry demonstrated that systemic hypothermia reduced whole-brain temperature, whereas FH primarily reduced the temperature of the ischemic region. CONCLUSIONS: Focal brain hypothermia requires longer cooling periods to achieve the same protective efficacy as SH. However, FH mainly affects the ischemic region, and therefore represents a promising and nonstressful alternative to SH.