Intraventricular hemorrhage induces inflammatory brain damage with blood-brain barrier dysfunction in immature rats.

BACKGROUND: We aimed to characterize a preclinical model of intraventricular hemorrhage-induced brain damage (IVH-BD) in extremely low birth weight newborns (ELBWN), to identify potential therapeutic targets based on its pathophysiology. METHODS: IVH was induced in 1-day-old (P1) Wistar rats by left periventricular injection of clostridium collagenase (PVCC). At P6, P14, and P45 IVH-BD (area of damage, motor and cognitive deficits, Lactate/N-acetylaspartate ratio), white matter injury (WMI: ipsilateral hemisphere and corpus callosum atrophy, oligodendroglial population and myelin basic protein signal reduction), blood-brain barrier (BBB) dysfunction (occludin and Mfsd2a expression, Gadolinium leakage) and inflammation (TNFα, TLR4, NFkB, and MMP9 expression; immune cell infiltration), excitotoxicity (Glutamate/N-acetylaspartate), and oxidative stress (protein nitrosylation) were assessed. Sham animals were similarly studied. RESULTS: IVH-BD leads to long-term WMI, resulting in motor and cognitive impairment, thus reproducing IVH-BD features in ELBWN. BBB dysfunction with increased permeability was observed at P6 and P14, coincident with an increased inflammatory response with TLR4 overexpression, increased TNFα production, and increased immune cell infiltration, as well as increased excitotoxicity and oxidative stress. CONCLUSIONS: This model reproduced some key hallmarks of IVH-BD in ELBWN. Inflammation associated with BBB dysfunction appears as relevant therapeutic target to prevent IVH-BD-induced WMI. IMPACT: Paraventricular injection of clostridium collagenase (PVCC) to 1-day-old Wistar rats uniquely reproduced the neuroimaging, histologic and functional characteristics of intraventricular hemorrhage-induced brain damage (IVH-BD) in extremely low birth weight newborns (ELBWN). PVCC-induced IVH triggered a prolonged inflammatory response associated with blood-brain barrier increased permeability, which in turn facilitates the infiltration of inflammatory cells. Thus, PVCC led to white matter injury (WMI) resulting in long-term motor and cognitive impairment. This model offers a valuable tool to obtain further insight into the mechanisms of IVH-BD in ELBWN and proposes some key therapeutic targets.

Localization of the cannabinoid type-1 receptor in subcellular astrocyte compartments of mutant mouse hippocampus.

Astroglial type-1 cannabinoid (CB1 ) receptors are involved in synaptic transmission, plasticity and behavior by interfering with the so-called tripartite synapse formed by pre- and post-synaptic neuronal elements and surrounding astrocyte processes. However, little is known concerning the subcellular distribution of astroglial CB1 receptors. In particular, brain CB1 receptors are mostly localized at cells' plasmalemma, but recent evidence indicates their functional presence in mitochondrial membranes. Whether CB1 receptors are present in astroglial mitochondria has remained unknown. To investigate this issue, we included conditional knock-out mice lacking astroglial CB1 receptor expression specifically in glial fibrillary acidic protein (GFAP)-containing astrocytes (GFAP-CB1 -KO mice) and also generated genetic rescue mice to re-express CB1 receptors exclusively in astrocytes (GFAP-CB1 -RS). To better identify astroglial structures by immunoelectron microscopy, global CB1 knock-out (CB1 -KO) mice and wild-type (CB1 -WT) littermates were intra-hippocampally injected with an adeno-associated virus expressing humanized renilla green fluorescent protein (hrGFP) under the control of human GFAP promoter to generate GFAPhrGFP-CB1 -KO and -WT mice, respectively. Furthermore, double immunogold (for CB1 ) and immunoperoxidase (for GFAP or hrGFP) revealed that CB1 receptors are present in astroglial mitochondria from different hippocampal regions of CB1 -WT, GFAP-CB1 -RS and GFAPhrGFP-CB1 -WT mice. Only non-specific gold particles were detected in mouse hippocampi lacking CB1 receptors. Altogether, we demonstrated the existence of a precise molecular architecture of the CB1 receptor in astrocytes that will have to be taken into account in evaluating the functional activity of cannabinergic signaling at the tripartite synapse.

Cannabidiol reduces intraventricular hemorrhage brain damage, preserving myelination and preventing blood brain barrier dysfunction in immature rats.

Intraventricular hemorrhage (IVH) is an important cause of long-term disability in extremely preterm infants, with no current treatment. This study assessed the potential neuroprotective effects of cannabidiol (CBD) in an IVH model using immature rats. IVH was induced in 1-day-old (P1) Wistar rats by left periventricular injection of Clostridial collagenase. Some rats received CBD prenatally (10 â€‹mg/kg i.p. to the dam) and then 5 â€‹mg/kg i.p. 6, 30 and 54 â€‹h after IVH (IVH+CBD, n â€‹= â€‹30). Other IVH rats received vehicle (IVH+VEH, n â€‹= â€‹34) and vehicle-treated non-IVH rats served as controls (SHM, n â€‹= â€‹29). Rats were humanely killed at P6, P14 or P45. Brain damage (motor and memory performance, area of damage, Lactate/N-acetylaspartate ratio), white matter injury (ipsilateral hemisphere and corpus callosum volume, oligodendroglial cell density and myelin basic protein signal), blood-brain barrier (BBB) integrity (Mfsd2a, occludin and MMP9 expression, gadolinium leakage), inflammation (TLR4, NFκB and TNFα expression, infiltration of pro-inflammatory cells), excitotoxicity (Glutamate/N-acetylspartate ratio) and oxidative stress (protein nitrosylation) were then evaluated. CBD prevented the long-lasting motor and cognitive consequences of IVH, reduced brain damage in the short- and long-term, protected oligodendroglial cells preserving adequate myelination and maintained BBB integrity. The protective effects of CBD were associated with the modulation of inflammation, excitotoxicity and oxidative stress. In conclusion, in immature rats, CBD reduced IVH-induced brain damage and its short- and long-term consequences, showing robust and pleiotropic neuroprotective effects. CBD is a potential candidate to ameliorate IVH-induced immature brain damage.

Increased expression of cannabinoid CB2 and serotonin 5-HT1A heteroreceptor complexes in a model of newborn hypoxic-ischemic brain damage.

Preclinical work shows cannabidiol as a promising drug to manage neonatal hypoxic-ischemic brain damage (NHIBD). The molecular mechanism is not well defined but the beneficial effects of this phytocannabinoid are blocked by antagonists of both cannabinoid CB2 (CB2R) and serotonin 5-HT1A (5-HT1AR) receptors that, in addition, may form heteromers in a heterologous expression system. Using bioluminescence energy transfer, we have shown a direct interaction of the two receptors that leads to a particular signaling in a heterologous system. A property attributed to the heteromer, namely cross-antagonism, was found in primary cultures of neurons thus indicating the occurrence of the receptor heteromer in the CNS. Oxygen-glucose deprivation to neurons led to an increase of CB2R-mediated signaling and an upregulation of CB2-5-HT1A heteroreceptor complex expression. In situ proximity ligation assays in brain cortical sections were performed to compare the expression of CB2-5-HT1A complexes in rat E20 fetuses and at different postnatal days. The expression, which is elevated in fetus and shortly after birth, was sharply reduced at later ages (even at P7). The expression of heteromer receptors was more marked in a model of NHIBD and, remarkably, the drop in expression was significantly delayed with respect to controls. These results indicate that CB2-5-HT1A heteroreceptor complex may be considered as a target in the therapy of the NHIBD. This article is part of the Special Issue entitled 'Receptor heteromers and their allosteric receptor-receptor interactions'.

Neuroprotection by cannabidiol and hypothermia in a piglet model of newborn hypoxic-ischemic brain damage.

OBJECTIVE: Hypothermia, the gold standard after a hypoxic-ischemic insult, is not beneficial in all treated newborns. Cannabidiol is neuroprotective in animal models of newborn hypoxic-ischemic encephalopathy. This study compared the relative efficacies of cannabidiol and hypothermia in newborn hypoxic-ischemic piglets and assessed whether addition of cannabidiol augments hypothermic neuroprotection. METHODS: One day-old HI (carotid clamp and FiO2 10% for 20 min) piglets were randomized to vehicle or cannabidiol 1 mg/kg i.v. u.i.d. for three doses after being submitted to normothermia or 48 h-long hypothermia with a subsequent rewarming period of 6 h. Non-manipulated piglets (naïve) served as controls. Hemodynamic or respiratory parameters as well as brain activity (aEEG amplitude) were monitored throughout the experiment. Following termination, brains were obtained for histological (TUNEL staining, apoptosis; immunohistochemistry for Iba-1, microglia), biochemical (protein carbonylation, oxidative stress; and TNFα concentration, neuroinflammation) or proton magnetic resonance spectroscopy (Lac/NAA: metabolic derangement; Glu/NAA: excitotoxicity). RESULTS: HI led to sustained depressed brain activity and increased microglial activation, which was significantly improved by cannabidiol alone or with hypothermia but not by hypothermia alone. Hypoxic-ischemic-induced increases in Lac/NAA, Glu/NAA, TNFα or apoptosis were not reversed by either hypothermia or cannabidiol alone, but combination of the therapies did. No treatment modified the effects of HI on oxidative stress or astroglial activation. Cannabidiol treatment was well tolerated. CONCLUSIONS: cannabidiol administration after hypoxia-ischemia in piglets offers some neuroprotective effects but the combination of cannabidiol and hypothermia shows some additive effect leading to more complete neuroprotection than cannabidiol or hypothermia alone.

Changes in the Stoichiometry of Uniplex Decrease Mitochondrial Calcium Overload and Contribute to Tolerance of Cardiac Ischemia/Reperfusion Injury in Hypothyroidism.

Background: Thyroid hormone status in hypothyroidism (HT) downregulates key elements in Ca2+ handling within the heart, reducing contractility, impairing the basal energetic balance, and increasing the risk of cardiovascular disease. Mitochondrial Ca2+ transport is reduced in HT, and tolerance to reperfusion damage has been documented, but the precise mechanism is not well understood. Therefore, we aimed to determine the stoichiometry and activity of the mitochondrial Ca2+ uniporter or uniplex in an HT model and the relevance to the opening of the mitochondrial permeability transition pores (mPTP) during ischemia/reperfusion (I/R) injury. Methods: An HT model was established in Wistar rats by treatment with 6-propylthiouracil for 28 days. Uniplex composition and activity were determined in cardiac mitochondria. Hearts were perfused ex vivo to induce I/R injury, and functional parameters related to contractility and tissue viability were evaluated. Results: The cardiac stoichiometry between two subunits of the uniplex (MICU1/MCU) increased by 25% in animals with HT. The intramitochondrial Ca2+ content was reduced by 40% and was less prone to the mPTP opening. After I/R injury, ischemic contracture and the onset of ventricular fibrillation were delayed in animals with HT, concomitant with a reduction in oxidative damage and mitochondrial dysfunction. Conclusions: Our results suggest that HT is associated with an increase in the cardiac MICU1/MCU ratio, thereby changing the stoichiometry between these subunits to increase the threshold to cytosolic Ca2+ and reduce mitochondrial Ca2+ overload. Our results also demonstrate that this HT model can be used to explore the role of mitochondrial Ca2+ transport in cardiac diseases due to its induced tolerance to cardiac damage.

Cannabinoid signalling in the immature brain: Encephalopathies and neurodevelopmental disorders.

The endocannabinoid system exerts a crucial neuromodulatory role in many brain areas that is essential for proper regulation of neuronal activity. The role of cannabinoid signalling controlling neuronal activity in the adult brain is also evident when considering its contribution to adult brain insults or neurodegenerative diseases. In the context of brain genetic or acquired encephalopathies administration of cannabinoid-based molecules has demonstrated to exert symptomatic relief and hence, they are proposed as new potential therapeutic compounds. This review article summarizes the main evidences indicating the beneficial action of cannabinoid-derived molecules in preclinical models of neonatal hypoxia/ischemic damage. In a second part, we discuss the available evidences of therapeutic actions of cannabidiol in children with refractory epilepsy syndromes. Finally, we discuss the current view of cannabinoid signalling mechanisms active in the immature brain that affect in neural cell fate and can contribute to long-term neural cell plasticity.

Anatomical characterization of the cannabinoid CB1 receptor in cell-type-specific mutant mouse rescue models.

Type 1 cannabinoid (CB1 ) receptors are widely distributed in the brain. Their physiological roles depend on their distribution pattern, which differs remarkably among cell types. Hence, subcellular compartments with little but functionally relevant CB1 receptors can be overlooked, fostering an incomplete mapping. To overcome this, knockin mice with cell-type-specific rescue of CB1 receptors have emerged as excellent tools for investigating CB1 receptors' cell-type-specific localization and sufficient functional role with no bias. However, to know whether these rescue mice maintain endogenous CB1 receptor expression level, detailed anatomical studies are necessary. The subcellular distribution of hippocampal CB1 receptors of rescue mice that express the gene exclusively in dorsal telencephalic glutamatergic neurons (Glu-CB1 -RS) or GABAergic neurons (GABA-CB1 -RS) was studied by immunoelectron microscopy. Results were compared with conditional CB1 receptor knockout lines. As expected, CB1 immunoparticles appeared at presynaptic plasmalemma, making asymmetric and symmetric synapses. In the hippocampal CA1 stratum radiatum, the values of the CB1 receptor-immunopositive excitatory and inhibitory synapses were Glu-CB1 -RS, 21.89% (glutamatergic terminals); 2.38% (GABAergic terminals); GABA-CB1 -RS, 1.92% (glutamatergic terminals); 77.92% (GABAergic terminals). The proportion of CB1 receptor-immunopositive excitatory and inhibitory synapses in the inner one-third of the dentate molecular layer was Glu-CB1 -RS, 53.19% (glutamatergic terminals); 2.30% (GABAergic terminals); GABA-CB1 -RS, 3.19% (glutamatergic terminals); 85.07% (GABAergic terminals). Taken together, Glu-CB1 -RS and GABA-CB1 -RS mice show the usual CB1 receptor distribution and expression in hippocampal cell types with specific rescue of the receptor, thus being ideal for in-depth anatomical and functional investigations of the endocannabinoid system. J. Comp. Neurol. 525:302-318, 2017. © 2016 Wiley Periodicals, Inc.

Differential Control of Cocaine Self-Administration by GABAergic and Glutamatergic CB1 Cannabinoid Receptors.

The type 1 cannabinoid receptor (CB1) modulates numerous neurobehavioral processes and is therefore explored as a target for the treatment of several mental and neurological diseases. However, previous studies have investigated CB1 by targeting it globally, regardless of its two main neuronal localizations on glutamatergic and GABAergic neurons. In the context of cocaine addiction this lack of selectivity is critical since glutamatergic and GABAergic neuronal transmission is involved in different aspects of the disease. To determine whether CB1 exerts different control on cocaine seeking according to its two main neuronal localizations, we used mutant mice with deleted CB1 in cortical glutamatergic neurons (Glu-CB1) or in forebrain GABAergic neurons (GABA-CB1). In Glu-CB1, gene deletion concerns the dorsal telencephalon, including neocortex, paleocortex, archicortex, hippocampal formation and the cortical portions of the amygdala. In GABA-CB1, it concerns several cortical and non-cortical areas including the dorsal striatum, nucleus accumbens, thalamic, and hypothalamic nuclei. We tested complementary components of cocaine self-administration, separating the influence of primary and conditioned effects. Mechanisms underlying each phenotype were explored using in vivo microdialysis and ex vivo electrophysiology. We show that CB1 expression in forebrain GABAergic neurons controls mouse sensitivity to cocaine, while CB1 expression in cortical glutamatergic neurons controls associative learning processes. In accordance, in the nucleus accumbens, GABA-CB1 receptors control cocaine-induced dopamine release and Glu-CB1 receptors control AMPAR/NMDAR ratio; a marker of synaptic plasticity. Our findings demonstrate a critical distinction of the altered balance of Glu-CB1 and GABA-CB1 activity that could participate in the vulnerability to cocaine abuse and addiction. Moreover, these novel insights advance our understanding of CB1 neuropathophysiology.

Alteraciones histopatológicas causadas por la roya Puccinia nakanishikii (Pucciniales: Pucciniaceae) en plantas de Cymbopogon citratus (Poaceae) / Histopathological alterations caused by Puccinia nakanishikii rust (Pucciniales: Pucciniaceae) in Cymbopogon citratus (Poaceae) plants

Introducción: Los aspectos histopatológicos e histoquímicos relacionados con el ataque de royas en plantas, así como su relación con los diferentes estados espóricos, son escasos en la literatura científica. Objetivos: Describir y analizar los aspectos histopatológicos e histoquímicos en Cymbopogon citratus y su relación con los diferentes estados espóricos de la roya Puccinia nakanishikii. Métodos: Durante abril y agosto 2013 se recolectaron hojas sanas e infectadas con Puccinia nakanishikii en la escarpa noroccidental de la meseta de Bucaramanga-Colombia. Las muestras con lesiones en diferentes etapas del desarrollo se fijaron y procesaron de acuerdo a protocolos estándar para la inclusión y corte en parafina y resina. Las secciones obtenidas en parafina (5-7 µm) fueron teñidas con Safranina-azul de Alcian y azul de Alcian-Hematoxilina. En tanto que las secciones obtenidas en resina (0.5 µm) se tiñeron con azul de Toluidina. También se elaboraron secciones a mano alzada para análisis de autofluorescencia. Las observaciones y registro fotográfico se efectuaron con microscopio fotónico y microscopía de epifluorescencia. Para observaciones con microscopía electrónica de barrido (MEB), las muestras se fijaron en Glutaraldehído, se deshidrataron con 2,2 dimetoxipropano, se desecaron a punto crítico y se metalizaron con oro. Resultados: Las hojas son por lo general hipostomáticas, con células epidérmicas largas y cortas formando filas paralelas y con la presencia de tricomas unicelulares espinosos y microtricomas. La superficie abaxial está cubierta por una densa capa de ceras epicuticulares y la adaxial está formada por agrupaciones de células buliformes y células epidérmicas de contorno rectangular o cuadrado. En el mesófilo no hay diferenciación entre parénquima de empalizada y esponjoso y su anatomía refleja el metabolismo C4 presente en esta gramínea. Se observó la formación de urediosoros y teliosoros hipófilos. Las urediosporas son la fase de reinfección y estas tienen de 4-5 poros germinativos ecuatoriales y su pared es equinulada. Las teliosporas son de pared lisa y de pedicelo persistente. Las urediosporas forman tubos de geminación por lo general sobre la superficie abaxial de la hoja y se desarrollan en dirección de los estomas, por donde penetran al interior del mesófilo. No se observó la presencia de apresorios. La epidermis se desprende y levanta por del desarrollo de las urediosporas y las paráfisis capitadas, a medida que el urediosoro crece. Con el avance de la infección, los tejidos fotosintéticos se desorganizan, pierden la autofluorescencia de la clorofila y las células sufren necrosis. Posteriormente, los tejidos vasculares se fragmentan y colapsan. Para este momento, la infección se ha extendido sobre toda la lámina foliar llevando a la muerte de la hoja y defoliación de la planta. Durante etapas avanzadas de la infección en los urediosoros se observaron picnidios, probablemente del hiperparásito Sphaerellopsis, asociados estrechamente a los tejidos infectados por la roya. Conclusiones: Puccinia nakanishikii se desarrolla sobre las hojas de Cymbopogon citratus produciendo urediosoros y teliosoros. Las urediosporas son la fase de reinfección, y las teliosporas solo se observaron en etapas avanzadas de la infección. La epidermis y los tejidos fotosintéticos son severamente afectados por la necrosis celular. En etapas avanzadas de la infección los tejidos vasculares se ven afectados.

Introduction: Histopathological and histochemical aspects linked to the attack of fungal rusts to plants, as well as its relation with the different spore stages are topics rather scarce in the scientific literature. Objective: To describe and analyze the histopathological and histochemical aspects of Cymbopogon citratus and its relation with the different stages of the spores from the rust fungi Puccinia nakanishikii. Methods: During the months April and August 2013, leaves healthy and infected by Puccinia nakanishikii were collected in the Northwestern scarp of the Bucaramanga-Colombia plateau. The samples with injuries on diverse developmental stages were fixated and processed according to the standard protocols for embedding and sectioning in paraffin and resin. Sections obtained from paraffin (5-7 µm) were stained with Safranin-Alcian blue and Alcian blue-Hematoxylin. On the other hand, sections obtained from resin (0.5 µm) were stained with Toluidine blue. Further, freehand sections were obtained for an autofluorescence analysis. The observations and photographic record were done via photonic microscope and epifluorescence microscope. For the observations via scanning electron microscopy (SEM), the samples were fixated in Glutaraldehyde, dehydrated with 2,2 dimethoxypropane, then desiccated to critical point and finally coated with gold. Results: The leaves are generally hypostomatic, with long and short epidermic cell forming parallel rows and showing unicellular prickle trichomes and micro-trichomes. The abaxial surface is covered by epicuticular wax forming a dense layer. The adaxial epidermis is formed by groupings of bulliform cells and epidermal cells with rectangular or squared contour. In the mesophyll, there is no differentiation between palisade and spongy parenchyma, its anatomy reflects the C4 metabolism. The formation of uredosori and teliosori both hypophyllous was observed. Urediniospores are the reinfecting agents phase, they have 4-5 equatorial germ pores and echinulate wall. Teliospores have smooth wall and a persistent pedicel. The urediniospores form a germ tube, generally on the abaxial leaf surface, these tubes develop towards the stomata reaching the mesophyll interior. No appressorium were observed. The epidermis limiting the uredosorus detaches due the development and pressure that exert both the urediniospores and capitate paraphyses. As the infection progresses, autofluorescense of the chlorophyll is lost and the cells undergo necrotic processes. Afterwards, the phloem collapses and the xylem becomes slightly disorganized. At this moment, the infection is extended along the whole leaf blade, resulting in the leaf death and the plant defoliation. On advanced stages of the infection, the uredosori showed pycnidia, probably belonging to the hyperparasite Sphaerellopsis, these structures were closely associated to the rust infected tissues. Conclusions: Puccinia nakanishikii develops on the leaves of Cymbopogon citratus producing uredosori and teliosori. Urediniospores are the reinfective stage, teliospores were only observed at late stages of the infection. The epidermis and photosynthetic tissue are severely affected by cell necrosis. The vascular tissues are deeply affected on the advances stages of the infection.

Cachexia assessed by bioimpedance vector analysis as a prognostic indicator in chronic stable heart failure patients.

BACKGROUND: This study explored whether the cachectic state assessed by bioimpedance vector analysis provides additional prognostic information about mortality from all causes. METHODS: We included 519 consecutive patients with stable chronic heart failure (mean age 62.5 ± 16.4 y; 286 males). Cachexia was identified in those subjects who fell outside the right lower quadrant of the reference curve of 95% on the resistance/reactance graph [bioelectrical impedance vectorial analysis (BIVA)-cachexia]. Clinical, anthropometric, and biochemical data were also evaluated. RESULTS: Patients with BIVA-cachexia (n = 196, 37.8%) were older and had significantly lower ejection fraction, handgrip strength, serum albumin, total cholesterol, and triglycerides. The frequency of patients with body mass index < 20, decreased muscle strength, hypoalbuminemia, anemia, anorexia, New York Heart Association functional classes III/IV and edema, as well as creatinine levels, resistance/height, and impedance index was significantly higher in the cachexia group. During 29 ± 11 mo of follow-up, 39 (19.9%) patients with BIVA-cachexia and 38 (11.7%) patients without BIVA-cachexia (P < 0.0001) died. CONCLUSIONS: The cachectic state is an independent risk factor for mortality in chronic heart failure patients. BIVA could represent a valuable tool to assess presence of cachexia as changes in body cell mass in heart failure patients because provide information additional to weight loss.