Dual-Hit: Glyphosate exposure at NOAEL level negatively impacts birth and glia-behavioural measures in heterozygous shank3 mutants.

The omnipresence of environmental contaminants represents a health danger with ramifications for adverse neurological trajectories. Here, we tested the dual-hit hypothesis that continuous exposure to non-observable adverse effect level (NOAEL) glyphosate from pre-natal to adulthood represents a risk factor for neurological-associated adaptations when in the presence of the heterozygote or homozygote mutation of the Shank3 synaptic gene. Ultrasound analysis of pregnant dams revealed patterns of pre-natal mortality with effects dependent on wild-type, Shank3ΔC/+, or Shank3ΔC/ΔC genotypes exposed to NOAEL glyphosate (GLY) compared to unexposed conditions. The postnatal survival rate was negatively impacted, specifically in Shank3ΔC/+ exposed to GLY. Next, the resulting six groups of pups were tracked into adulthood and analyzed for signs of neuroinflammation and neurological adaptions. Sholl's analysis revealed cortical microgliosis across groups exposed to GLY, with Shank3ΔC/+ mice presenting the most significant modifications. Brain tissues were devoid of astrocytosis, except for the perivascular compartment in the cortex in response to GLY. Distinct behavioral adaptations accompanied these cellular modifications, as locomotion and social preference were decreased in Shank3ΔC/+ mice exposed to GLY. Notably, GLY exposure from weaning did not elicit glial or neurological adaptations across groups, indicating the importance of pre-natal contaminant exposure. These results unveil the intersection between continuous pre-natal to adulthood environmental input and a pre-existing synaptic mutation. In an animal model, NOAEL GLY predominantly impacted Shank3ΔC/+ mice, compounding an otherwise mild phenotype compared to Shank3ΔC/ΔC. The possible relevance of these findings to neurodevelopmental risk is critically discussed, along with avenues for future research.

Neurovascular hypoxia after mild traumatic brain injury in juvenile mice correlates with heart-brain dysfunctions in adulthood.

AIM: Retrospective studies suggest that mild traumatic brain injury (mTBI) in pediatric patients may lead to an increased risk of cardiac events. However, the exact functional and temporal dynamics and the associations between heart and brain pathophysiological trajectories are not understood. METHODS: A single impact to the left somatosensory cortical area of the intact skull was performed on juvenile mice (17 days postnatal). Cerebral 3D photoacoustic imaging was used to measure the oxygen saturation (sO2 ) in the impacted area 4 h after mTBI followed by 2D and 4D echocardiography at days 7, 30, 90, and 190 post-impact. At 8 months, we performed a dobutamine stress test to evaluate cardiac function. Lastly, behavioral analyses were conducted 1 year after initial injury. RESULTS: We report a rapid and transient decrease in cerebrovascular sO2 and increased hemoglobin in the impacted left brain cortex. Cardiac analyses showed long-term diastolic dysfunction and a diminished systolic strain response under stress in the mTBI group. At the molecular level, cardiac T-p38MAPK and troponin I expression was pathologic modified post-mTBI. We found linear correlations between brain sO2 measured immediately post-mTBI and long-term cardiac strain after 8 months. We report that initial cerebrovascular hypoxia and chronic cardiac dysfunction correlated with long-term behavioral changes hinting at anxiety-like and memory maladaptation. CONCLUSION: Experimental juvenile mTBI induces time-dependent cardiac dysfunction that corresponds to the initial neurovascular sO2 dip and is associated with long-term behavioral modifications. These imaging biomarkers of the heart-brain axis could be applied to improve clinical pediatric mTBI management.

Significance of developmental meningeal lymphatic dysfunction in experimental post-traumatic injury.

Understanding the pathological mechanisms unfolding after chronic traumatic brain injury (TBI) could reveal new therapeutic entry points. During the post-TBI sequel, the involvement of cerebrospinal fluid drainage through the meningeal lymphatic vessels was proposed. Here, we used K14-VEGFR3-Ig transgenic mice to analyze whether a developmental dysfunction of meningeal lymphatic vessels modifies post-TBI pathology. To this end, a moderate TBI was delivered by controlled cortical injury over the temporal lobe in male transgenic mice or their littermate controls. We performed MRI and a battery of behavioral tests over time to define the post-TBI trajectories. In vivo analyses were integrated by ex-vivo quantitative and morphometric examinations of the cortical lesion and glial cells. In post-TBI K14-VEGFR3-Ig mice, the recovery from motor deficits was protracted compared to littermates. This outcome is coherent with the observed slower hematoma clearance in transgenic mice during the first two weeks post-TBI. No other genotype-related behavioral differences were observed, and the volume of cortical lesions imaged by MRI in vivo, and confirmed by histology ex-vivo, were comparable in both groups. However, at the cellular level, post-TBI K14-VEGFR3-Ig mice exhibited an increased percentage of activated Iba1 microglia in the hippocampus and auditory cortex, areas that are proximal to the lesion. Although not impacting or modifying the structural brain damage and post-TBI behavior, a pre-existing dysfunction of meningeal lymphatic vessels is associated with morphological microglial activation over time, possibly representing a sub-clinical pathological imprint or a vulnerability factor. Our findings suggest that pre-existing mLV deficits could represent a possible risk factor for the overall outcome of TBI pathology.

Seizure activity triggers tau hyperphosphorylation and amyloidogenic pathways.

OBJECTIVE: Although epilepsies and neurodegenerative disorders show pathophysiological similarities, their direct functional associations are unclear. Here, we tested the hypothesis that experimental seizures can induce tau hyperphosphorylation and amyloidogenic modifications over time, with intersections with neuroinflammation. METHODS: We used a model of mesial temporal lobe epilepsy (MTLE) where unilateral intrahippocampal injection of kainic acid (KA) in C57BL/6 mice elicits epileptogenesis and spontaneous focal seizures. We used a model of generalized status epilepticus (SE) obtained by intraperitoneal KA injection in C57BL/6 mice. We performed analyses and cross-comparisons according to a schedule of 72 h, 1 week, and 8 weeks after KA injection. RESULTS: In experimental MTLE, we show AT100, PHF1, and CP13 tau hyperphosphorylation during epileptogenesis (72 h-1 week) and long-term (8 weeks) during spontaneous seizures in the ipsilateral hippocampi, the epileptogenic zone. These pathological modifications extended to the contralateral hippocampus, a seizure propagating zone with no histological lesion or sclerosis. Two kinases, Cdk5 and GSK3ß, implicated in the pathological phosphorylation of tau, were activated. In this MTLE model, the induction of the amyloidogenic pathway (APP, C99, BACE1) was prominent and long-lasting in the epileptogenic zone. These Alzheimer's disease (AD)-relevant markers, established during seizure progression and recurrence, reciprocated an enduring glial (GFAP, Iba1) inflammation and the inadequate activation of the endogenous, anti-inflammatory, glucocorticoid receptor system. By contrast, a generalized SE episode provoked a predominantly transient induction of tau hyperphosphorylation and amyloidogenic markers in the hippocampus, along with resolving inflammation. Finally, we identified overlapping profiles of long-term hippocampal tau hyperphosphorylation by comparing MTLE to J20 mice, the latter a model relevant to AD. SIGNIFICANCE: MTLE and a generalized SE prompt persistent and varying tau hyperphosphorylation or amyloidogenic modifications in the hippocampus. In MTLE, an AD-relevant molecular trajectory intertwines with neuroinflammation, spatiotemporally involving epileptogenic and nonlesional seizure propagating zones.

Neurovascular multiparametric MRI defines epileptogenic and seizure propagation regions in experimental mesiotemporal lobe epilepsy.

OBJECTIVE: Improving the identification of the epileptogenic zone and associated seizure-spreading regions represents a significant challenge. Innovative brain-imaging modalities tracking neurovascular dynamics during seizures may provide new disease biomarkers. METHODS: With use of a multi-parametric magnetic resonance imaging (MRI) analysis at 9.4 Tesla, we examined, elaborated, and combined multiple cellular and cerebrovascular MRI read-outs as imaging biomarkers of the epileptogenic and seizure-propagating regions. Analyses were performed in an experimental model of mesial temporal lobe epilepsy (MTLE) generated by unilateral intra-hippocampal injection of kainic acid (KA). RESULTS: In the ipsilateral epileptogenic hippocampi, tissue T1 and blood-brain barrier (BBB) permeability to gadolinium were increased 48-72 hours post-KA, as compared to sham and contralateral hippocampi. BBB permeability endured during spontaneous focal seizures (4-6 weeks), along with a significant increase of apparent diffusion coefficient (ADC) and blood volume fraction (BVf). Simultaneously, ADC and BVf were augmented in the contralateral hippocampus, a region characterized by electroencephalographic seizure spreading, discrete histological neurovascular cell modifications, and no tissue sclerosis. We next asked whether combining all the acquired MRI parameters could deliver criteria to classify the epileptogenic from the seizure-spreading and sham hippocampi in these experimental conditions and over time. To differentiate sham from epileptogenic areas, the automatic multi-parametric classification provided a maximum accuracy of 97.5% (32 regions) 48-72 hours post-KA and of 100% (60 regions) at spontaneous seizures stage. To differentiate sham, epileptogenic, and seizure-spreading areas, the accuracies of the automatic classification were 93.1% (42 regions) 48-72 hours post-KA and 95% (80 regions) at spontaneous seizure stage. SIGNIFICANCE: Combining multi-parametric MRI acquisition and machine-learning analyses delivers specific imaging identifiers to segregate the epileptogenic from the contralateral seizure-spreading hippocampi in experimental MTLE. The potential clinical value of our findings is critically discussed.

Varying modalities of perinatal exposure to a pesticide cocktail elicit neurological adaptations in mice and zebrafish.

Epidemiological indications connect maternal and developmental presence or exposure to pesticides with an increased risk for a spectrum of neurological trajectories. To provide pre-clinical data in support of this hypothesis, we used two distinct experimental models. First, female and male mice were fed immediately prior to mating, and the resulting pregnant dams were continously fed during gestation and lactation periods using chow pellets containing a cocktail of six pesticides at tolerable daily intake levels. Male and female offspring were then tracked for behavioral and in vivo electrophysiological adaptations. Second, a zebrafish model allowed us to screen toxicity and motor-behavior outcomes specifically associated with the developmental exposure to a low-to-high concentration range of the cocktail and of each individual pesticide. Here, we report anxiety-like behavior in aging male mice maternally exposed to the cocktail, as compared to age and gender matched sham animals. In parallel, in vivo electrocorticography revealed a decrease in gamma (40-80 Hz) and an increase of theta (6-9 Hz) waves, delineating a long-term, age-dependent, neuronal slowing. Neurological changes were not accompanied by brain structural malformations. Next, by using zebrafish larvae, we showed an increase of all motor-behavioral parameters resulting from the developmental exposure to 10 µg/L of pesticide cocktail, an outcome that was not associated with midbrain structural or neurovascular modifications as assessed by in vivo 2-photon microscopy. When screening each pesticide, chlorpyrifos elicited modifications of swimming parameters at 0.1 µg/L, while other components provoked changes from 0.5 µg/L. Ziram was the single most toxic component inducing developmental malformations and mortality at 10 µg/L. Although we have employed non-equivalent modalities and timing of exposure in two dissimilar experimental models, these outcomes indicate that presence of a pesticide cocktail during perinatal periods represents an element promoting behavioral and neurophysiological modifications. The study limitations and the possible pertinence of our findings to ecotoxicology and public health are critically discussed.

Machine Learning Analysis of the Cerebrovascular Thrombi Proteome in Human Ischemic Stroke: An Exploratory Study.

Objective: Mechanical retrieval of thrombotic material from acute ischemic stroke patients provides a unique entry point for translational research investigations. Here, we resolved the proteomes of cardioembolic and atherothrombotic cerebrovascular human thrombi and applied an artificial intelligence routine to examine protein signatures between the two selected groups. Methods: We specifically used n = 32 cardioembolic and n = 28 atherothrombotic diagnosed thrombi from patients suffering from acute stroke and treated by mechanical thrombectomy. Thrombi proteins were successfully separated by gel-electrophoresis. For each thrombi, peptide samples were analyzed by nano-flow liquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS) to obtain specific proteomes. Relative protein quantification was performed using a label-free LFQ algorithm and all dataset were analyzed using a support-vector-machine (SVM) learning method. Data are available via ProteomeXchange with identifier PXD020398. Clinical data were also analyzed using SVM, alone or in combination with the proteomes. Results: A total of 2,455 proteins were identified by nano-LC-MS/MS in the samples analyzed, with 438 proteins constantly detected in all samples. SVM analysis of LFQ proteomic data delivered combinations of three proteins achieving a maximum of 88.3% for correct classification of the cardioembolic and atherothrombotic samples in our cohort. The coagulation factor XIII appeared in all of the SVM protein trios, associating with cardioembolic thrombi. A combined SVM analysis of the LFQ proteome and clinical data did not deliver a better discriminatory score as compared to the proteome only. Conclusion: Our results advance the portrayal of the human cerebrovascular thrombi proteome. The exploratory SVM analysis outlined sets of proteins for a proof-of-principle characterization of our cohort cardioembolic and atherothrombotic samples. The integrated analysis proposed herein could be further developed and retested on a larger patients population to better understand stroke origin and the associated cerebrovascular pathophysiology.

Life-long Dietary Pesticide Cocktail Induces Astrogliosis Along with Behavioral Adaptations and Activates p450 Metabolic Pathways.

Exposure to environmental contaminants is a public health concern. However, pre-clinical studies that examine the impact of pesticides at low-dose and the long-term consequences are uncommon. Here, C57BL6/j male and female mice were daily fed from weaning and up to 12 months, corresponding to early-childhood into middle-age in humans, using chow pellets containing a cocktail of pesticides at tolerable daily intake levels. We found that 12 months of dietary exposure to pesticides was associated with a moderate perenchymal or perivascular astrogliosis in specific hippocampal sub-regions. The expression of platelet-derived growth factor receptor beta was modified at the perivascular level. Examination of Iba1+ microglial cells did not reveal sizeable changes. Concomitantly to astrogliosis, spontaneous spatial memory and sociability were modified in males at 12 months of dietary exposure to pesticides. Telemetry electrocorticograhic explorations ruled out the presence of epileptiform activity or theta-gamma wave modifications in these conditions. Long-term pesticides impacted the periphery where the hepatic P450 metabolic cytochromes Cyp4a14 and Cyp4a10 were significantly upregulated in male and female mice during the 12 months of exposure. The expression of ß-oxidation genes, such as Acox1, Cpt1a and Eci, was also significantly increased in male and female mice in response to pesticides. Collectively, our results indicate that a life-long exposure to a pesticide cocktail elicits sex-dependent, spatio-temporally restricted brain modifications and significant activation of P450 pathways in the periphery. These brain-peripheral adjustments are discussed as time or age-dependent vulnerability elements.

Early cerebrovascular and long-term neurological modifications ensue following juvenile mild traumatic brain injury in male mice.

Clinical evidence suggests that a mild traumatic brain injury occurring at a juvenile age (jmTBI) may be sufficient to elicit pathophysiological modifications. However, clinical reports are not adequately integrated with experimental studies examining brain changes occurring post-jmTBI. We monitored the cerebrovascular modifications and assessed the long-term behavioral and electrographic changes resulting from experimental jmTBI. In vivo photoacoustic imaging demonstrated a decrease of cerebrovascular oxygen saturation levels in the impacted area hours post-jmTBI. Three days post-jmTBI oxygenation returned to pre-jmTBI levels, stabilizing at 7 and 30 days after the injury. At the functional level, cortical arterioles displayed no NMDA vasodilation response, while vasoconstriction induced by thromboxane receptor agonist was enhanced at 1 day post-jmTBI. Arterioles showed abnormal NMDA vasodilation at 3 days post-jmTBI, returning to normality at 7 days post injury. Histology showed changes in vessel diameters from 1 to 30 days post-jmTBI. Neurological evaluation indicated signs of anxiety-like behavior up to 30 days post-jmTBI. EEG recordings performed at the cortical site of impact 30 days post-jmTBI did not indicate seizures activity, although it revealed a reduction of gamma waves as compared to age matched sham. Histology showed decrease of neuronal filament staining. In conclusion, experimental jmTBI triggers an early cerebrovascular hypo­oxygenation in vivo and faulty vascular reactivity. The exact topographical coherence and the direct casualty between early cerebrovascular changes and the observed long-term neurological modifications remain to be investigated. A potential translational value for cerebro-vascular oxygen monitoring in jmTBI is discussed.

Experimental Myocardial Infarction Elicits Time-Dependent Patterns of Vascular Hypoxia in Peripheral Organs and in the Brain.

Aims: Microvascular alterations occurring after myocardial infarction (MI) may represent a risk factor for multi-organ failure. Here we used in vivo photoacoustic (PA) imaging to track and define the changes in vascular oxygen saturation (sO2) occurring over time after experimental MI in multiple peripheral organs and in the brain. Methods and Results: Experimental MI was obtained in BALB/c mice by permanent ligation of the left anterior descending artery. PA imaging (Vevo LAZR-X) allowed tracking mouse-specific sO2 kinetics in the cardiac left ventricular (LV) anterior wall, brain, kidney, and liver at 4 h, 1 day, and 7 days post-MI. Here we reported a correlation between LV sO2 and longitudinal anterior myocardial strain after MI (r = -0.44, p < 0.0001, n = 96). Acute LV dysfunction was associated with global hypoxia, specifically a decrease in sO2 level in the brain (-5.9%), kidney (-6.4%), and liver (-7.3%) at 4 and 24 h post-MI. Concomitantly, a preliminary examination of capillary NG2DsRed pericytes indicated cell rarefication in the heart and kidney. While the cardiac tissue was persistently impacted, sO2 levels returned to pre-MI levels in the brain and in peripheral organs 7 days after MI. Conclusions: Collectively, our data indicate that experimental MI elicits precise trajectories of vascular hypoxia in peripheral organs and in the brain. PA imaging enabled the synchronous tracking of oxygenation in multiple organs and occurring post-MI, potentially enabling a translational diagnostic modality for the identification of vascular modifications in this disease setting.

The GR-ANXA1 pathway is a pathological player and a candidate target in epilepsy.

Immune changes occur in experimental and clinical epilepsy. Here, we tested the hypothesis that during epileptogenesis and spontaneous recurrent seizures (SRS) an impairment of the endogenous anti-inflammatory pathway glucocorticoid receptor (GR)-annexin A1 (ANXA1) occurs. By administrating exogenous ANXA1, we studied whether pharmacological potentiation of the anti-inflammatory response modifies seizure activity and pathophysiology. We used an in vivo model of temporal lobe epilepsy based on intrahippocampal kainic acid (KA) injection. Video-electroencephalography, molecular biology analyses on brain and peripheral blood samples, and pharmacological investigations were performed in this model. Human epileptic cortices presenting type II focal cortical dysplasia (IIa and b), hippocampi with or without hippocampal sclerosis (HS), and available controls were used to study ANXA1 expression. A decrease of phosphorylated (phospho-) GR and phospho-GR/tot-GR protein expression occurred in the hippocampus during epileptogenesis. Downstream to GR, the anti-inflammatory protein ANXA1 remained at baseline levels while inflammation installed and endured. In peripheral blood, ANXA1 and corticosterone levels showed no significant modifications during disease progression except for an early and transient increase poststatus epilepticus. These results indicate inadequate ANXA1 engagement over time and in these experimental conditions. By analyzing human brain specimens, we found that where significant inflammation exists, the pattern of ANXA1 immunoreactivity was abnormal because the typical perivascular ANXA1 immunoreactivity was reduced. We next asked whether potentiation of the endogenous anti-inflammatory mechanism by ANXA1 administration modifies the disease pathophysiology. Although with varying efficacy, administration of exogenous ANXA1 somewhat reduced the time spent in seizure activity as compared to saline. These results indicate that the anti-inflammatory GR-ANXA1 pathway is defective during experimental seizure progression. The prospect of pharmacologically restoring or potentiating this endogenous anti-inflammatory mechanism as an add-on therapeutic strategy for specific forms of epilepsy is proposed.-Zub, E., Canet, G., Garbelli, R., Blaquiere, M., Rossini, L., Pastori, C., Sheikh, M., Reutelingsperger, C., Klement, W., de Bock, F., Audinat, E., Givalois, L., Solito, E., Marchi, N. The GR-ANXA1 pathway is a pathological player and a candidate target in epilepsy.

A pericyte-glia scarring develops at the leaky capillaries in the hippocampus during seizure activity.

OBJECTIVE: Inflammatory cerebrovascular damage occurs in epilepsy. Here, we tested the hypothesis that a pericyte-glia scar forms around the outer wall of hippocampal capillaries in a model of temporal lobe epilepsy associated with hippocampal sclerosis. We studied the participation of stromal cells expressing platelet-derived growth factor receptor beta (PDGFRß) and extracellular matrix modifications to the perivascular scar during epileptogenesis. METHODS: We used NG2DsRed/C57BL6 mice and induced status epilepticus (SE) followed by epileptogenesis and spontaneous recurrent seizures (SRS) by means of unilateral intrahippocampal injection of kainic acid (KA). For pharmacological assessment, we used organotypic hippocampal cultures (OHCs) where ictal electrographic activity was elicited by KA or bicuculline. RESULTS: NG2DsRed pericytes, GFAP astroglia, and IBA1 microglia are reactive and converge to form a pericapillary multicellular scar in the CA hippocampal regions during epileptogenesis and at SRS. The capillaries are leaky as indicated by fluorescein entering the parenchyma from the peripheral blood. Concomitantly, PDGFRß transcript and protein levels were significantly increased. Within the regional scar, a fibrotic-like PDGFRß mesh developed around the capillaries, peaking at 1 week post-SE and regressing, but not resolving, at SRS. Abnormal distribution or accumulation of extracellular matrix collagens III/IV occurred in the CA regions during seizure progression. PDGFRß/DAPI cells were in direct contact with or adjacent to the damaged NG2DsRed pericytes at the capillary interface, consistent with the notion of stromal cell reactivity or fibroblast formation. Inducing electrographic activity in OHCs was sufficient to augment PDGFRß reactivity around the capillaries. The latter effect was pharmacologically mimicked by treating OHCs with the PDGFRß agonist PDGF-BB and it was diminished by the PDGFRß inhibitor imatinib. SIGNIFICANCE: The reported multicellular activation and scar are traits of perivascular inflammation and hippocampal sclerosis in experimental epilepsy, with an implication for neurovascular dysfunction. Modulation of PDGFRß could be exploited to target inflammation in this chronic disease setting.

Seizure progression and inflammatory mediators promote pericytosis and pericyte-microglia clustering at the cerebrovasculature.

BACKGROUND: Cerebrovascular dysfunction and inflammation occur in epilepsy. Here we asked whether pericytes, a pivotal cellular component of brain capillaries, undergo pathological modifications during experimental epileptogenesis and in human epilepsy. We evaluated whether pro-inflammatory cytokines, present in the brain during seizures, contribute to pericyte morphological modifications. METHODS: In vivo, unilateral intra-hippocampal kainic acid (KA) injections were performed in NG2DsRed/C57BL6 mice to induce status epilepticus (SE), epileptogenesis, and spontaneous recurrent seizures (SRS). NG2DsRed mice were used to visualize pericytes during seizure progression. The effect triggered by recombinant IL-1ß, TNFα, or IL-6 on pericytes was evaluated in NG2DsRed hippocampal slices and in human-derived cell culture. Human brain specimens obtained from temporal lobe epilepsy (TLE) with or without sclerosis (HS) and focal cortical dysplasia (FCD-IIb) were evaluated for pericyte-microglial cerebrovascular assembly. RESULTS: A disarray of NG2DsRed+ pericyte soma and ramifications was found 72 h post-SE and 1 week post-SE (epileptogenesis) in the hippocampus. Pericyte modifications topographically overlapped with IBA1+ microglia clustering around the capillaries with cases of pericytes lodged within the microglial cells. Microglial clustering around the NG2DsRed pericytes lingered at SRS. Pericyte proliferation (Ki67+) occurred 72 h post-SE and during epileptogenesis and returned towards control levels at SRS. Human epileptic brain tissues showed pericyte-microglia assemblies with IBA1/HLA microglial cells outlining the capillary wall in TLE-HS and FCD-IIb specimens. Inflammatory mediators contributed to pericyte modifications, in particular IL-1ß elicited pericyte morphological changes and pericyte-microglia clustering in NG2DsRed hippocampal slices. Modifications also occurred when pro-inflammatory cytokines were added to an in vitro culture of pericytes. CONCLUSIONS: These results indicate the occurrence of pericytosis during seizures and introduce a pericyte-microglial mediated mechanism of blood-brain barrier dysfunction in epilepsy.