Microglia through MFG-E8 signaling decrease the density of degenerating neurons and protect the brain from the development of cortical infarction after stroke.

Neuronal loss is a hallmark of stroke and other neurodegenerative diseases, and as such, neuronal loss caused by microglia has been thought to be a contributing factor to disease progression. Here, we show that microglia indeed contribute significantly to neuronal loss in a mouse model of stroke, but this microglial-dependent process of neuronal clearance specifically targets stressed and degenerating neurons in the ischemic cortical region and not healthy non-ischemic neurons. Nonspecific stimulation of microglia decreased the density of neurons in the ischemic cortical region, whereas specific inhibition of MFG-E8 signaling, which is required for microglial phagocytosis of neurons, had the opposite effect.

TAT peptide at treatment-level concentrations crossed brain endothelial cell monolayer independent of receptor-mediated endocytosis or peptide-inflicted barrier disruption.

The peptide domain extending from residues 49 to 57 of the HIV-1 Tat protein (TAT) has been widely shown to facilitate cell entry of and blood-brain barrier (BBB) permeability to covalently bound macromolecules; therefore, TAT-linked therapeutic peptides trafficked through peripheral routes have been used to treat brain diseases in preclinical and clinical studies. Although the mechanisms underlying cell entry by similar peptides have been established to be temperature-dependent and cell-type specific and to involve receptor-mediated endocytosis, how these peptides cross the BBB remains unclear. Here, using an in vitro model, we studied the permeability of TAT, which was covalently bound to the fluorescent probe fluorescein isothiocyanate (FITC), and evaluated whether it crossed the "in vitro BBB", a monolayer of brain endothelial cells, and whether the mechanisms were similar to those involved in TAT entry into cells.

Chronic Visual Stimulation with LED Light Flickering at 24, 40, or 80 Hz Failed to Reduce Amyloid β Load in the 5XFAD Alzheimer's Disease Mouse Model.

A single 1-h session (or 7 d of daily 1-h sessions) of noninvasive visual stimulation with LED light flickering at 40 Hz, but not at 20 or 80 Hz, was reported to increase microglial size and decrease amyloid β (Aβ) load in the 5xFAD mouse model of Alzheimer's disease. To achieve better therapeutic benefits, we explored the effects of daily 1-h sessions of visual stimulation with continuous light or LED light flickering at 24, 40, or 80 Hz for a period of five weeks in 5xFAD mice. As expected, 33-week-old 5xFAD mice but not control wild-type mice of the same age exhibited an abundance of swollen microglia and Aβ plaques in the visual cortex and hippocampus.

Blood-Brain Barrier Disruption in Preclinical Mouse Models of Stroke Can Be an Experimental Artifact Caused by Craniectomy.

The pathophysiological features of ischemia-related blood-brain barrier (BBB) disruption are widely studied using preclinical stroke models. However, in many of these models, craniectomy is required to confirm arterial occlusion via laser Doppler flowmetry or to enable direct ligation of the cerebral artery. In the present study, mice were used to construct a distal middle cerebral artery occlusion (dMCAO) model, a preclinical stroke model that requires craniectomy to enable direct ligation of the cerebral artery, or were subjected to craniectomy alone.

Drug-Induced Hyperglycemia as a Potential Contributor to Translational Failure of Uncompetitive NMDA Receptor Antagonists.

Hyperglycemia is a comorbidity in 60-80% of stroke patients; nevertheless, neuroprotective drugs like NMDA receptor (NMDAR) antagonists are typically assessed in normoglycemic animals at the preclinical stage before they are approved to enter clinical trials. Interestingly, as a possible explanation for the translational failure of NMDAR antagonists, it was recently reported that stroke occurring during nighttime causes smaller infarctions in rodents and therefore has a smaller window for neuroprotection. To investigate why stroke occurring during different circadian phases confers a difference in severity, we reanalyzed the published source data and found that some mice that were used in the daytime have higher blood glucose than mice that were used in the nighttime.

Stroke injury induced by distal middle cerebral artery occlusion is resistant to N-methyl-d-aspartate receptor antagonism in FVB/NJ mice.

Although N-methyl-d-aspartate receptor (NMDAR) antagonism has been shown to have a neuroprotective effect in many preclinical stroke models, the efficacy of this antiexcitotoxicity strategy in clinical trials in stroke patients has been disappointing. Interestingly, it has been reported that NMDAR antagonism is not neuroprotective in C57BL/6 mice subjected to distal middle cerebral artery occlusion (dMCAO), supporting the notion that whether these treatments are neuroprotective depends on the type of cerebral ischemia. However, because C57BL/6 mice are inherently resistant to excitotoxicity, the reported lack of neuroprotection could also be explained by the difference in the mouse strain studied rather than the stroke model used.

Evans blue dye as an indicator of albumin permeability across a brain endothelial cell monolayer in vitro.

An increase in the brain endothelial (BEnd) cell permeability of blood albumin is often seen as an early sign of blood-brain barrier (BBB) disruption and can precede increases in the BEnd permeability of small molecules and other plasma proteins in the course of brain disease. Therefore, Evans blue dye (EBD), an albumin-binding fluorescent tracer that is simple to detect and quantify, has been widely utilized for studying BEnd permeability during BBB disruption. Here, we investigated whether EBD is a suitable indicator of albumin permeability across mouse BEnd cell monolayers, alone or cocultured with mouse cortical astrocytes, in an in-vitro permeability assay; given the strong affinity of EBD for albumin, we further asked whether EBD can affect albumin permeability and vice versa.

Citric Acid in Drug Formulations Causes Pain by Potentiating Acid-Sensing Ion Channel 1.

Pain at the injection site is a common complaint of patients receiving therapeutic formulations containing citric acid. Despite the widely acknowledged role of acid-sensing ion channels (ASICs) in acid-related perception, the specific ASIC subtype mediating pain caused by subcutaneous acid injection and the mechanism by which citrate affects this process are less clear. Here, male mice subjected to intraplantar acid injection responded by executing a withdrawal reflex, and this response was abolished by ASIC1 but not ASIC2 knockout.

Microglia depletion by PLX3397 has no effect on cocaine-induced behavioral sensitization in male mice.

Cocaine and other addictive drugs are known to stimulate microglia, and microglia in turn have been shown to play roles in both the development and mitigation of drug dependence. For instance, cocaine can directly bind to surface receptors on microglia and trigger their release of interleukin-1β, which promotes addictive behaviors; however, cocaine also indirectly stimulates microglia by elevating dopamine, which causes microglia to impair long-lasting neuronal changes related to cocaine use. The seemingly opposing roles of microglia beg the question of what the net effect of microglial presence is on cocaine-induced behavioral changes.

Native expression of ASIC1a and ASIC1b human homologues in the HEK 293 cell line allows pharmacological evaluation of analgesics targeting acid sensation in humans.

Nociceptors arising from the dorsal root ganglia (DRG) express acid-sensing ion channel-1 (ASIC1) subtypes to mediate the perception of inflammatory and neuropathic pain, and as such, these receptors are attractive targets for the development of analgesics for these painful conditions. Nevertheless, given that the human and rodent DRG differ considerably in subtype proportions of ASIC1 and that the pharmacological properties of rodent ASIC1 subtypes and their human homologues are distinct, ASIC1 inhibitors that demonstrate analgesic properties in rodents may not necessarily be effective in preventing pain in humans. In this study, we show that human embryonic kidney (HEK) 293 cells, which are routinely used as a cellular vehicle for the heterologous expression and pharmacological characterization of receptors and ion channels, natively transcribe the human homologues of ASIC1a and ASIC1b at similar proportions to those found in the human DRG.

Hypothermia but not NMDA receptor antagonism protects against stroke induced by distal middle cerebral arterial occlusion in mice.

Excitotoxicity mediated by the N-methyl-D-aspartate receptor (NMDAR) is believed to be a primary mechanism of neuronal injury following stroke. Thus, many drugs and therapeutic peptides were developed to inhibit either the NMDAR at the cell surface or its downstream intracellular death-signaling cascades. Nevertheless, the majority of focal ischemia studies concerning NMDAR antagonism were performed using the intraluminal suture-induced middle cerebral arterial occlusion (MCAO) model, which produces a large cortical and subcortical infarct leading to hypothalamic damage and fever in experimental animals.

Isoflurane attenuates carbogen-induced blood-brain barrier disruption independent of body temperature in mice and rats.

Isoflurane protects the blood-brain barrier (BBB) against cerebral extravasation of Evans blue dye (EBD), a commonly used serum protein tracer, in animals subjected to BBB disruption. As such, it has been implicated as a therapeutic agent that can prevent brain edema and damage caused by a number of brain insults, including focal ischemia and subarachnoid hemorrhage. Recently, it has been shown that isoflurane inhibits the cerebral extravasation of EBD following ischemic stroke chiefly by inducing hypothermia, raising the intriguing possibility that isoflurane protected against other causes of BBB disruption also through hypothermia.

Carbogen inhalation opens the blood-brain barrier in rats without causing long-term metabolic or neurological deficit.

The blood-brain barrier (BBB) prevents many drugs from entering the brain. Yet, conventional methods that open the BBB are technically demanding, poorly reversible, and can be associated with long-term adverse effects. In comparison, carbogen, which is introduced nearly a century ago as a treatment for psychiatric disorders, is easy to administer and readily available to many labs and hospitals.

Vascular delivery of intraperitoneal Evans blue dye into the blood-brain barrier-intact and disrupted rat brains.

Blood-brain barrier (BBB) integrity can be determined by tracer infusion into the circulation followed by measurements of its penetration into the brain parenchyma. Tracer injection through the intraperitoneal (i.p.

Anesthesia-Induced Hypothermia Attenuates Early-Phase Blood-Brain Barrier Disruption but Not Infarct Volume following Cerebral Ischemia.

Blood-brain barrier (BBB) disruption is thought to facilitate the development of cerebral infarction after a stroke. In a typical stroke model (such as the one used in this study), the early phase of BBB disruption reaches a peak 6 h post-ischemia and largely recovers after 8-24 h, whereas the late phase of BBB disruption begins 48-58 h post-ischemia. Because cerebral infarct develops within 24 h after the onset of ischemia, and several therapeutic agents have been shown to reduce the infarct volume when administered at 6 h post-ischemia, we hypothesized that attenuating BBB disruption at its peak (6 h post-ischemia) can also decrease the infarct volume measured at 24 h.

Adenosine receptor agonist NECA increases cerebral extravasation of fluorescein and low molecular weight dextran independent of blood-brain barrier modulation.

Conventional methods for therapeutic blood-brain barrier (BBB) disruption facilitate drug delivery but are cumbersome to perform. A previous study demonstrated that adenosine receptor (AR) stimulation by 5'-N-ethylcarboxamide adenosine (NECA) increased the extravasation of intravascular tracers into the brain and proposed that AR agonism may be an effective method for therapeutic BBB disruption. We attempted to confirm the extravasation of tracers into the brain and also investigated tracer extravasation into peripheral organs and tracer retention in the blood.

Optimization of Evans blue quantitation in limited rat tissue samples.

Evans blue dye (EBD) is an inert tracer that measures plasma volume in human subjects and vascular permeability in animal models. Quantitation of EBD can be difficult when dye concentration in the sample is limited, such as when extravasated dye is measured in the blood-brain barrier (BBB) intact brain. The procedure described here used a very small volume (30 µl) per sample replicate, which enabled high-throughput measurements of the EBD concentration based on a standard 96-well plate reader.

Excitotoxicity and stroke: identifying novel targets for neuroprotection.

Excitotoxicity, the specific type of neurotoxicity mediated by glutamate, may be the missing link between ischemia and neuronal death, and intervening the mechanistic steps that lead to excitotoxicity can prevent stroke damage. Interest in excitotoxicity began fifty years ago when monosodium glutamate was found to be neurotoxic. Evidence soon demonstrated that glutamate is not only the primary excitatory neurotransmitter in the adult brain, but also a critical transmitter for signaling neurons to degenerate following stroke.

Distinct patterns of cerebral extravasation by Evans blue and sodium fluorescein in rats.

The Evans blue dye (EBD; 961 Da) and the sodium fluorescein dye (NaF; 376 Da) are commonly used inert tracers in blood-brain barrier (BBB) research. They are both highly charged low molecular weight (LMW) tracers with similar lipophobic profiles. Nevertheless, the EBD binds to serum albumin (69,000 Da) to become a high molecular weight (HMW) protein tracer when injected into the circulation, whereas the NaF remains an unbound small molecule in the circulation.

Role of stress-inducible protein-1 in recruitment of bone marrow derived cells into the ischemic brains.

Stress-inducible protein-1 (STI-1) is the proposed ligand for the cellular prion protein (PrP(C) ), which is thought to facilitate recovery following stroke. Whether STI-1 expression is affected by stroke and how its signalling facilitates recovery remain elusive. Brain slices from patients that died of ischemic stroke were collected for STI-1 immunohistochemistry.

Molecular level activation insights from a NR2A/NR2B agonist.

N-methyl D-aspartate receptors (NMDARs), a subclass of glutamate receptors have broad actions in neural transmission for major brain functions. Overactivation of NMDARs leading to "excitotoxicity" is the underlying mechanism of neuronal death in a number of neurological diseases, especially stroke. Much research effort has been directed toward developing pharmacological agents to modulate NMDAR actions for treating neurological diseases, in particular stroke.

Intravenous mannitol does not increase blood-brain barrier permeability to inert dyes in the adult rat forebrain.

Intravenous mannitol (IV-M) is widely administered in the clinic to lower intracranial pressure in patients with brain trauma and stroke. However, intracarotid arterial mannitol (ICA-M) is known to potently open the blood-brain barrier (BBB) to serum protein tracers such as the Evans blue dye (EBD). In this study, we aimed to determine the potential effect of IV-M on BBB permeability to EBD and a small molecular tracer sodium fluorescein dye (NaF).

Increase in Evans blue dye extravasation into the brain in the late developmental stage.

The development of the blood-brain barrier (BBB) against permeability to inert tracers, such as Evans blue dye (EBD), occurs quite early on at embryonic stages (before E13-E15), and the BBB remains resistant to EBD between E15 and early adulthood (P20-P30). Here, we aimed to examine the changes in EBD permeability at a later stage in development, specifically comparing young rats (P20) with adult rats (P86). We found markedly higher EBD extravasation into the forebrains of adult rats compared with those of the young rats (P=0.

Stroke intervention pathways: NMDA receptors and beyond.

Despite abundant evidence from basic/preclinical research that excessive NMDAR (N-methyl-d-aspartate receptor) stimulation is a crucial step required for brain damage following a stroke, clinical trials for NMDAR blockers have all ended with disappointments. The past decade of stroke research has revealed distinct NMDAR subpopulations and many specific effectors downstream of these receptors that are differentially responsible for neuronal survival and death. These new advancements provide promising targets for the development of novel NMDAR-based neuroprotective stroke therapies that could have greater therapeutic windows and reduced side effects.