Cell permeable HMGB1-binding heptamer peptide ameliorates neurovascular complications associated with thrombolytic therapy in rats with transient ischemic stroke.

BACKGROUND: Blood-brain barrier (BBB) breakdown and inflammatory responses are the major causes of tissue-type plasminogen activator (tPA)-induced hemorrhagic transformation (HT), while high-mobility group box 1 (HMGB1) exacerbates inflammatory damage to BBB during the process of brain ischemia/reperfusion. This study aimed to investigate the change of HMGB1 after thrombolytic therapy and whether blocking HMGB1 could ameliorate the neurovasculature complications secondary to tPA treatment in stroke rats. METHODS: Sera from acute stroke patients and rats with thrombolytic therapy were collected to investigate HMGB1 secretion. Male Sprague-Dawley rats with 2 h or 4.5 h middle cerebral artery occlusion were continuously infused with tPA followed by administration of membrane permeable HMGB1-binding heptamer peptide (HBHP). The mortality rate, neurological score, HT, brain swelling, BBB permeability, and inflammatory factors were determined. RESULTS: The results revealed that HMGB1 levels were elevated in both stroke patients and rats after tPA treatment. Blocking HMGB1 signaling by HBHP in the rat model of 4.5 h brain ischemia significantly attenuated tPA-related complications, including mortality rate, the degree of hemorrhage, brain swelling, neurological deficits, BBB impairment, microglia activation, and the expressions of inflammatory cytokines. CONCLUSIONS: tPA treatment might induce HMGB1 secretion while blocking HMGB1 with HBHP could markedly reduce the risk of thrombolysis-associated brain hemorrhage and mortality through attenuating BBB damage and inflammatory reactions. These results indicate that HMGB1 may potentiate the risk of HT in tPA administration and that blocking HMGB1 signaling would be helpful in preventing complications brought by thrombolysis in ischemic stroke. TRIAL REGISTRATION: http://www.chictr.org.cn . Unique identifier: ChiCTR-OOC-16010052. Registered 30 November 2016.

Representation of conspecific vocalizations in amygdala of awake marmosets.

Human speech and animal vocalizations are important for social communication and animal survival. Neurons in the auditory pathway are responsive to a range of sounds, from elementary sound features to complex acoustic sounds. For social communication, responses to distinct patterns of vocalization are usually highly specific to an individual conspecific call, in some species. This includes the specificity of sound patterns and embedded biological information. We conducted single-unit recordings in the amygdala of awake marmosets and presented calls used in marmoset communication, calls of other species and calls from specific marmoset individuals. We found that some neurons (47/262) in the amygdala distinguished 'Phee' calls from vocalizations of other animals and other types of marmoset vocalizations. Interestingly, a subset of Phee-responsive neurons (22/47) also exhibited selectivity to one out of the three Phees from two different 'caller' marmosets. Our findings suggest that, while it has traditionally been considered the key structure in the limbic system, the amygdala also represents a critical stage of socially relevant auditory perceptual processing.

Induction of Anxiety-Like Phenotypes by Knockdown of Cannabinoid Type-1 Receptors in the Amygdala of Marmosets.

The amygdala is an important hub for regulating emotions and is involved in the pathophysiology of many mental diseases, such as depression and anxiety. Meanwhile, the endocannabinoid system plays a crucial role in regulating emotions and mainly functions through the cannabinoid type-1 receptor (CB1R), which is strongly expressed in the amygdala of non-human primates (NHPs). However, it remains largely unknown how the CB1Rs in the amygdala of NHPs regulate mental diseases. Here, we investigated the role of CB1R by knocking down the cannabinoid receptor 1 (CNR1) gene encoding CB1R in the amygdala of adult marmosets through regional delivery of AAV-SaCas9-gRNA. We found that CB1R knockdown in the amygdala induced anxiety-like behaviors, including disrupted night sleep, agitated psychomotor activity in new environments, and reduced social desire. Moreover, marmosets with CB1R-knockdown had up-regulated plasma cortisol levels. These results indicate that the knockdown of CB1Rs in the amygdala induces anxiety-like behaviors in marmosets, and this may be the mechanism underlying the regulation of anxiety by CB1Rs in the amygdala of NHPs.

Hand-Rearing Method for Infant Marmosets.

The common marmoset (Callithrix jacchus) is a small and highly social New World monkey with high reproduction rates, which has been proven to be a compelling non-human primate model for biomedical and neuroscience research. Some females give birth to triplets; however, the parents cannot raise all of them. To save these infants, we have developed a hand-rearing method for raising newborn marmosets. In this protocol, we describe the formula of the food, the time for feeding, the configuration of the temperature and humidity, as well as the adaptation of the hand-reared infants to the colony environment. This hand-rearing method significantly increases the survival rate of marmoset infants (without hand-rearing: 45%; with hand-rearing: 86%) and provides the opportunity to study the development of marmoset infants with similar genetic backgrounds raised in different postnatal environments. As the method is practical and easy to use, we anticipate that it could also be applied to other labs working with common marmosets.

Deconstruction of Vermal Cerebellum in Ramp Locomotion in Mice.

The cerebellum is involved in encoding balance, posture, speed, and gravity during locomotion. However, most studies are carried out on flat surfaces, and little is known about cerebellar activity during free ambulation on slopes. Here, it has been imaged the neuronal activity of cerebellar molecular interneurons (MLIs) and Purkinje cells (PCs) using a miniaturized microscope while a mouse is walking on a slope. It has been found that the neuronal activity of vermal MLIs specifically enhanced during uphill and downhill locomotion. In addition, a subset of MLIs is activated during entire uphill or downhill positions on the slope and is modulated by the slope inclines. In contrast, PCs showed counter-balanced neuronal activity to MLIs, which reduced activity at the ramp peak. So, PCs may represent the ramp environment at the population level. In addition, chemogenetic inactivation of lobule V of the vermis impaired uphill locomotion. These results revealed a novel micro-circuit in the vermal cerebellum that regulates ambulatory behavior in 3D terrains.

DeepBhvTracking: A Novel Behavior Tracking Method for Laboratory Animals Based on Deep Learning.

Behavioral measurement and evaluation are broadly used to understand brain functions in neuroscience, especially for investigations of movement disorders, social deficits, and mental diseases. Numerous commercial software and open-source programs have been developed for tracking the movement of laboratory animals, allowing animal behavior to be analyzed digitally. In vivo optical imaging and electrophysiological recording in freely behaving animals are now widely used to understand neural functions in circuits. However, it is always a challenge to accurately track the movement of an animal under certain complex conditions due to uneven environment illumination, variations in animal models, and interference from recording devices and experimenters. To overcome these challenges, we have developed a strategy to track the movement of an animal by combining a deep learning technique, the You Only Look Once (YOLO) algorithm, with a background subtraction algorithm, a method we label DeepBhvTracking. In our method, we first train the detector using manually labeled images and a pretrained deep-learning neural network combined with YOLO, then generate bounding boxes of the targets using the trained detector, and finally track the center of the targets by calculating their centroid in the bounding box using background subtraction. Using DeepBhvTracking, the movement of animals can be tracked accurately in complex environments and can be used in different behavior paradigms and for different animal models. Therefore, DeepBhvTracking can be broadly used in studies of neuroscience, medicine, and machine learning algorithms.

Glimepiride and glibenclamide have comparable efficacy in treating acute ischemic stroke in mice.

Glibenclamide protects against ischemic injury in both preclinical and clinical studies, presumably by blocking the de novo assembled sulfonylurea receptor 1-transient receptor potential M4 (Sur1-Trpm4) channel induced by ischemia. However, glibenclamide may cause unexpected serious hypoglycemia. Here, we tested whether glimepiride, another sulfonylurea with better safety, has comparable efficacy with glibenclamide and whether gene deletion of Trpm4 (Trpm4-/-) exerts similar effect. Wild-type (WT) mice subjected to temporary middle cerebral artery occlusion (tMCAO) were randomized to receive glibenclamide (an initial dose of 10 µg/kg and additional doses of 1.2 µg every 8 h), three different doses of glimepiride (10 µg/kg, 100 µg/kg and 1 mg/kg) or vehicle after ischemia, while tMCAO-treated Trpm4-/- mice were randomized to receive vehicle or glimepiride. Neurological function, infarct volume, edema formation, the integrity of blood-brain barrier and inflammatory reaction were evaluated at 24 h after ischemia. In tMCAO-treated WT mice, 10 µg/kg and 100 µg/kg glimepiride had comparable efficacy with glibenclamide in improving longa score and grip test score, reducing infarct volume, mitigating brain edema, lessening extravasation of Evans blue dye and IgG, restoring tight junction protein expression as well as suppressing inflammatory cytokines. Compared with WT mice, Trpm4-/- mice showed less neurological deficit, smaller cerebral infarction, lighter brain edema and more integrity of blood-brain barrier. As expected, glimepiride did not provide additional neuroprotection compared with vehicle in the tMCAO-treated Trpm4-/- mice. Glimepiride shows comparable efficacy with glibenclamide in alleviating brain injury after ischemic stroke in mice, possibly via targeting the Sur1-Trpm4 channel.

Spinosin and 6'''­Feruloylspinosin protect the heart against acute myocardial ischemia and reperfusion in rats.

Investigating active compounds from Chinese herbal medicine that can rescue myocardial cells is a good approach to preserve cardiac function. Several herbal formulae that containing Semen Ziziphi Spinosae (SZS), also called Suanzaoren in Chinese, are clinically effective in the treatment of patients with acute myocardial infarction (AMI). The present study aimed to investigate the cardioprotective effects of spinosin and 6'''­feruloylspinosin, two flavonoid glycosides from SZS, in a rat model of myocardial ischemia and reperfusion. The left anterior descending artery (LAD) was occluded to induce myocardial ischemia. Spinosin or 6'''­feruloylspinosin (5 mg/kg) was intraperitoneally injected into rats 30 min before LAD ligation. The protein levels of myocardial enzymes in the serum, the extent of tissue injury and the rate of apoptosis were examined after AMI in rats with or without pretreatment with spinosin or 6'''­feruloylspinosin. Western blotting was performed to investigate the potential mechanisms underlying the function of these two flavonoid glycosides. The present results suggested that pretreatment with spinosin or 6'''­feruloylspinosin significantly attenuated myocardial tissue injury, and reduced myocardial enzyme release and cell apoptosis in AMI rats. In addition, spinosin treatment increased light chain 3B­II and 6'''­feruloylspinosin, and reduced p62, indicating that autophagy was promoted after drug treatments. Treatments of spinosin and 6'''­feruloylspinosin led to the reduction of glycogen synthase kinase­3ß (GSK3ß) phosphorylation at Tyr216, and the increase of peroxisome proliferator­activated receptor γ coactivator (PGC)­1α and its downstream signaling proteins, including nuclear factor (erythroid­derived 2)­like 2 (Nrf2) and hemeoxygenase1 (HO­1). The present data suggested that SZS flavonoids could protect myocardial cells against acute heart ischemia­reperfusion, probably via the inhibition of GSK3ß, which increased autophagy and the activity of the PGC­1α/Nrf2/HO­1 pathway.

Pathogenicity of an FAdV-4 isolate to chickens and its genomic analysis.

Fowl adenovirus serotype 4 (FAdV-4) strain SD1511 was isolated from chickens with severe inclusion body hepatitis and hydropericardium syndrome in Shandong Province, China. The isolate was cultured in primary chicken embryo kidney cells. A study of pathogenicity indicated that SD1511 readily infected 7-35-d-old chickens by intramuscular injection and intranasal and oral routes, causing 50%-100% mortality. The 35-d-old chickens suffered more severe infection than 7- and 21-d-old chickens with mortality highest in the intramuscular injection group. The serum from surviving chickens showed potent viral neutralizing capability. The complete genome of SD1511 was sequenced and analyzed. The strain was found to belong to the FAdV-4 cluster with more than 99% identity with the virulent FAdV-4 strains isolated in China in recent years except for some distinct variations, including deletions of open reading frame 27 (ORF27), ORF48, and part of ORF19. Our findings suggest that SD1511 might be used as a prototype strain for the study of pathogenesis and vaccine development.

IRAK-M Deficiency Exacerbates Ischemic Neurovascular Injuries in Experimental Stroke Mice.

Background: Innate immune response to neuronal death is one of the key events of the pathogenesis of ischemic brain injury. Interleukin-1 receptor-associated kinase (IRAK)-M, encoded by gene Irak3, negatively regulates toll-like receptor signaling by interacting with the MyD88-IRAK-4-IRAK-1 complex and blocking the phosphorylation and dissociation of IRAK-1. Its function in the ischemic stroke is unknown. Objective: This study aims to investigate whether IRAK-M deficiency could exacerbate neuroinflammation and neurovascular injuries during cerebral ischemia and reperfusion. Methods: Male C57BL/6 mice and Irak3 knockout mice were subjected to 45 min of middle cerebral artery occlusion and 4 or 24 h of reperfusion. Transcription of Irak3 gene was evaluated by quantitative real-time PCR (qRT-PCR). Then, infarct volume, neurological score, brain water content, and Evans blue leakage were compared between knock-out and wild-type mice after reperfusion. Through the observation of gross brain specimen after cerebral ischemia, the incidence of hemorrhage transformation was compared between KO and WT mice. To explore underlying signaling pathways involved in IRAK-M deficiency, major proinflammatory cytokines and NF-κB signaling were measured by qRT-PCR and Western blot. Results: The expression of IRAK-M peaked at 1 h after reperfusion, and then gradually decreased within the first 24 h, which was abolished by blocking the expression of hypoxia induced factor 1α. IRAK-M deficiency increased infarct volume, brain edema, the incidence of hemorrhage transformation, and the permeability of blood-brain barrier. In addition, the NF-κB-mediated expressions of proinflammatory cytokines and the activation of microglia in the ipsilateral brain from knock-out mice were much higher than those in wild-type littermates. Conclusion: IRAK-M deletion exacerbates neurovascular damages which are related to the pronounced activation of NF-κB signaling and neuroinflammatory responses during cerebral ischemia-reperfusion in mice. Our study indicates that IRAK-M has neuroprotective effect and has potential to facilitate the development of new pharmaceuticals that reduce neurovascular complications.

TLR4 signaling pathway mediates the LPS/ischemia-induced expression of monocytechemotactic protein-induced protein 1 in microglia.

Monocytechemotactic protein-induced protein 1 (MCPIP1), a newly recognized mRNA endonuclease, can be induced by lipopolysaccharide (LPS) and ischemic attack, then exerts a negative feedback loop against neuroinflammatory injury, but the specific underlying signaling pathway of the induction is unclear. Toll-like receptors (TLRs) and receptor for advanced glycation end products (RAGE) signaling pathways are involved in LPS/ischemia-evoked inflammation. This study aims to explore which receptor signaling is mainly involved in the induction of MCPIP1 by LPS and ischemic attack. BV2 cells and mice were subjected to LPS stimulation or transient middle cerebral artery occlusion (MCAO) to examine the modulation of MCPIP1. Specific inhibitors for TLR4, TLR2 or RAGE were preadministered to explore the mechanisms of MCPIP1 expression. Results showed that MCPIP1 was significantly increased by LPS and ischemic stress both in vitro and in vivo in time and dose dependent manners. Inhibition of TLR4, rather than TLR2 or RAGE, downregulated the LPS/ischemia-induced expression of MCPIP1 and reduced the levels of TLR4, MyD88, phosphorylated-MAPK (p-P38), phosphorylated-IκBα (p-IκBα), as well as the translocation of NF-κB (p65). In conclusion, we firstly demonstrate that TLR4 signaling pathway, not TLR2 or RAGE, predominantly mediates the LPS/ischemia-induced expression of MCPIP1 in microglia.