IFNγ-stimulated dendritic cell extracellular vesicles can be nasally administered to the brain and enter oligodendrocytes.

Extracellular vesicles secreted from IFNγ-stimulated rat dendritic cells (referred to here as IFNγ-DC-EVs) contain miRNAs which promote myelination (including but not limited to miR-219), and preferentially enter oligodendrocytes in brain slice cultures. IFNγ-DC-EVs also increase myelination when nasally administered to naïve rats. While we can infer that these extracellular vesicles enter the CNS from functional studies, here we demonstrate biodistribution throughout the brain after nasal delivery by way of imaging studies. After nasal administration, Xenolight DiR-labelled IFNγ-DC-EVs were detected 30 minutes later throughout the brain and the cervical spinal cord. We next examined cellular uptake of IFNγ-DC-EVs by transfecting IFNγ-DC-EVs with mCherry mRNA prior to nasal administration. mCherry-positive cells were found along the rostrocaudal axis of the brain to the brainstem. These cells morphologically resembled oligodendrocytes, and indeed cell-specific co-staining for neurons, astrocytes, microglia and oligodendrocytes showed that mcherry positive cells were predominantly oligodendrocytes. This is in keeping with our prior in vitro results showing that IFNγ-DC-EVs are preferentially taken up by oligodendrocytes, and to a lesser extent, microglia. To confirm that IFNγ-DC-EVs delivered cargo to oligodendrocytes, we quantified protein levels of miR-219 mRNA targets expressed in oligodendrocyte lineage cells, and found significantly reduced expression. Finally, we compared intranasal versus intravenous delivery of Xenolight DiR-labelled IFNγ-DC-EVs. Though labelled IFNγ-DC-EVs entered the CNS via both routes, we found that nasal delivery more specifically targeted the CNS with less accumulation in the liver. Taken together, these data show that intranasal administration is an effective route for delivery of IFNγ-DC-EVs to the CNS, and provides additional support for their development as an EV-based neurotherapeutic that, for the first time, targets oligodendrocytes.

Insulin-like growth factor-1 inhibits nitroglycerin-induced trigeminal activation of oxidative stress, calcitonin gene-related peptide and c-Fos expression.

Migraineurs experience increased oxidative stress which drives the initiation and maintenance of migraine-related pain in animal models and, by extension, migraine in humans. Oxidative stress augments calcitonin gene-related peptide (CGRP) levels, a mediator of migraine pain. Insulin-like growth factor-1 (IGF-1), a neuroprotective growth factor, reduces susceptibility to spreading depression, a preclinical model of migraine, in cultured brain slices by blocking oxidative stress and neuroinflammation from microglia. Similarly, nasal delivery of IGF-1 inhibits spreading depression in vivo. After recurrent cortical spreading depression, nasal administration of IGF-1 also significantly reduces trigeminal ganglion oxidative stress and CGRP levels as well as trigeminocervical c-Fos activation. Here, we probed for the impact of nasal IGF-1 pretreatment on trigeminal system activation using a second well-established preclinical model of migraine, systemic nitroglycerin injection. Adult male rats were treated with one of three doses of IGF-1 (37.5, 75 or 150 µg) and the optimal dose found in males was subsequently used for treatment of female rats. One day later, animals received an intraperitoneal injection of nitroglycerin. Measurements taken two hours later after nitroglycerin alone showed increased surrogate markers of trigeminal activation - oxidative stress and CGRP in the trigeminal ganglion and c-Fos in the trigeminocervical complex compared to vehicle control. These effects were significantly reduced at all doses of IGF-1 for trigeminal ganglion metrics of oxidative stress and CGRP and only at the lowest dose in both males and females for c-Fos. The latter inverted U-shaped or hormetic response is seen in enzyme-targeting drugs. While the specific mechanisms remain to be explored, our data here supports the ability of IGF-1 to preserve mitochondrial and antioxidant pathway homeostasis as means to prevent nociceptive activation in the trigeminal system produced by an experimental migraine model.

Insulin-like growth factor-1 inhibits spreading depression-induced trigeminal calcitonin gene related peptide, oxidative stress & neuronal activation in rat.

Migraineurs can show brain hyperexcitability and oxidative stress that may promote headache. Since hyperexcitability can enhance oxidative stress which promotes hyperexcitability, ending this feed-back loop may reduce migraine. Neocortical spreading depression, an animal model of migraine begins with hyperexcitability and triggers oxidative stress in the neocortical area involved and in the trigeminal system, which is important to pain pathway nociceptive activation in migraine. Additionally, oxidative stress causes increased trigeminal ganglion calcitonin gene-related peptide release and oxidative stress can reduce spreading depression threshold. Insulin-like growth factor-1 significantly protects against spreading depression in vitro by reducing oxidative stress and it is effective against spreading depression after intranasal delivery to animals. Here, we used adult male rats and extend this work to study the trigeminal system where insulin-like growth factor-1 receptors are highly expressed. Recurrent neocortical spreading depression significantly increased surrogate markers of trigeminal activation - immunostaining for trigeminal ganglion oxidative stress, calcitonin gene related peptide levels and c-fos in the trigeminocervical complex versus sham. These effects were significantly reduced by intranasal delivery of insulin-like growth factor-1 a day before recurrent neocortical spreading depression. Furthermore, intranasal treatment with insulin-like growth factor-1 significantly reduced naïve levels of trigeminal ganglion calcitonin gene related peptide versus sham with no impact on blood glucose levels. Intranasal delivery of insulin-like growth factor-1 not only mitigates neocortical spreading depression, a cause of migraine hyperexcitability modeled in animals, but also when neocortical spreading depression is triggered by supra-threshold stimuli, insulin-like growth factor-1 effectively reduces nociceptive activation in the trigeminal system.

IFNγ-Stimulated Dendritic Cell Exosomes for Treatment of Migraine Modeled Using Spreading Depression.

Migraine is a common headache disorder characterized by unilateral, intense headaches. In migraine with aura, the painful headache is preceded by focal neurological symptoms that can be visual, sensory, or motor in nature. Spreading depression (the most likely cause of migraine with aura and perhaps related headache pain) results in increased neuronal excitability and related increases in inflammation and production of reactive oxygen species. This in turn can promote the transformation of low-frequency, episodic migraine into higher-frequency and eventually chronic migraine. Though migraine affects 11% of adults worldwide, with 3% experiencing chronic headache, existing therapies offer only modest benefits. Here, we focus on the mechanisms by which environmental enrichment (i.e., volitionally increased intellectual, social, and physical activity) mitigates spreading depression. In prior work, we have shown that exposure to environmental enrichment reduces susceptibility to spreading depression in rats. This protective effect is at least in part due to environmental enrichment-mediated changes in the character of serum exosomes produced by circulating immune cells. We went on to show that environmental enrichment-mimetic exosomes can be produced by stimulating dendritic cells with low levels of interferon gamma (a cytokine that is phasically increased during environmental enrichment). Interferon gamma-stimulated dendritic cell exosomes (IFNγ-DC-Exos) significantly improve myelination and reduce oxidative stress when applied to hippocampal slice cultures. Here, we propose that they may also be effective against spreading depression. We found that administration of IFNγ-DC-Exos reduced susceptibility to spreading depression in vivo and in vitro, suggesting that IFNγ-DC-Exos may be a potential therapeutic for migraine.

Intranasally administered IGF-1 inhibits spreading depression in vivo.

Spreading depression (SD) is a wave of cellular depolarization that travels slowly through susceptible gray matter brain areas. SD is the most likely cause of migraine aura and perhaps migraine pain, and is a well-accepted animal model of migraine. Identification of therapeutics that can prevent SD may have clinical relevance toward migraine treatment. Here we show that insulin-like growth factor-1 (IGF-1) significantly inhibited neocortical SD in vivo after intranasal delivery to rats. A single dose of IGF-1 inhibited SD within an hour, and continued to protect for at least seven days thereafter. A two-week course of IGF-1, administered every third day, further decreased SD susceptibility and showed no aberrant effects on glial activation, nasal mucosa, or serum markers of toxicity. SD begets SD in vitro by mechanisms that involve microglial activation. We add to this relationship by showing that recurrent SD in vivo increased susceptibility to subsequent SD, and that intervention with IGF-1 significantly interrupted this pathology. These findings support nasal administration of IGF-1 as a novel intervention capable of mitigating SD susceptibility, and as a result, potentially migraine.

NIH workshop report on the trans-agency blood-brain interface workshop 2016: exploring key challenges and opportunities associated with the blood, brain and their interface.

A trans-agency workshop on the blood-brain interface (BBI), sponsored by the National Heart, Lung and Blood Institute, the National Cancer Institute and the Combat Casualty Care Research Program at the Department of Defense, was conducted in Bethesda MD on June 7-8, 2016. The workshop was structured into four sessions: (1) blood sciences; (2) exosome therapeutics; (3) next generation in vitro blood-brain barrier (BBB) models; and (4) BBB delivery and targeting. The first day of the workshop focused on the physiology of the blood and neuro-vascular unit, blood or biofluid-based molecular markers, extracellular vesicles associated with brain injury, and how these entities can be employed to better evaluate injury states and/or deliver therapeutics. The second day of the workshop focused on technical advances in in vitro models, BBB manipulations and nanoparticle-based drug carrier designs, with the goal of improving drug delivery to the central nervous system. The presentations and discussions underscored the role of the BBI in brain injury, as well as the role of the BBB as both a limiting factor and a potential conduit for drug delivery to the brain. At the conclusion of the meeting, the participants discussed challenges and opportunities confronting BBI translational researchers. In particular, the participants recommended using BBI translational research to stimulate advances in diagnostics, as well as targeted delivery approaches for detection and therapy of both brain injury and disease.

Environmental Enrichment Stimulates Immune Cell Secretion of Exosomes that Promote CNS Myelination and May Regulate Inflammation.

Environmental enrichment (EE) consists of increased physical, intellectual, and social activity, and has wide-ranging effects, including enhancing cognition, learning and memory, and motor coordination. Animal studies have demonstrated that EE improves outcome of brain trauma and neurodegenerative disorders, including demyelinating diseases like multiple sclerosis, making it a promising therapeutic option. However, the complexity of applying a robust EE paradigm makes clinical use difficult. A better understanding of the signaling involved in EE-based neuroprotection may allow for development of effective mimetics as an alternative. In prior work, we found that exosomes isolated from the serum of rats exposed to EE impact CNS myelination. Exosomes are naturally occurring nanovesicles containing mRNA, miRNA, and protein, which play important roles in cell function, disease, and immunomodulation. When applied to hippocampal slice cultures or nasally administered to naïve rats, EE-serum exosomes significantly increase myelin content, oligodendrocyte precursor (OPC) and neural stem cell levels, and reduce oxidative stress (OS). We found that rat EE exosomes were enriched in miR-219, which is necessary and sufficient for OPC differentiation into myelinating cells. Thus, peripherally produced exosomes may be a useful therapy for remyelination. Here, we aim to better characterize the impact of EE on CNS health and to determine the cellular source of nutritive exosomes found in serum. We found that exosomes isolated from various circulating immune cell types all increased slice culture myelin content, contained miR-219, and reduced OS, suggesting that EE globally alters immune function in a way that supports brain health.

Potential functional applications of extracellular vesicles: a report by the NIH Common Fund Extracellular RNA Communication Consortium.

The NIH Extracellular RNA Communication Program's initiative on clinical utility of extracellular RNAs and therapeutic agents and developing scalable technologies is reviewed here. Background information and details of the projects are presented. The work has focused on modulation of target cell fate by extracellular vesicles (EVs) and RNA. Work on plant-derived vesicles is of intense interest, and non-mammalian sources of vesicles may represent a very promising source for different therapeutic approaches. Retro-viral-like particles are intriguing. Clearly, EVs share pathways with the assembly machinery of several other viruses, including human endogenous retrovirals (HERVs), and this convergence may explain the observation of viral-like particles containing viral proteins and nucleic acid in EVs. Dramatic effect on regeneration of damaged bone marrow, renal, pulmonary and cardiovascular tissue is demonstrated and discussed. These studies show restoration of injured cell function and the importance of heterogeneity of different vesicle populations. The potential for neural regeneration is explored, and the capacity to promote and reverse neoplasia by EV exposure is described. The tremendous clinical potential of EVs underlies many of these projects, and the importance of regulatory issues and the necessity of general manufacturing production (GMP) studies for eventual clinical trials are emphasized. Clinical trials are already being pursued and should expand dramatically in the near future.

Phasic Treatment with Interferon Gamma Stimulates Release of Exosomes that Protect Against Spreading Depression.

The detrimental effects of T-cell-secreted interferon gamma (IFNγ) on oxidative stress (OS) and demyelination in multiple sclerosis (MS) are well recognized. Recently, we demonstrated that IFNγ-mediated damage to myelin also increases susceptibility to spreading depression (SD; the likely basis of migraine with aura). However, before onset of MS, induction of physiological levels of IFNγ, like that produced by environmental enrichment (EE), protects against demyelination and OS. Accordingly, we focused on the potential for physiological levels of IFNγ to protect against SD. EE, which occurs with a moderate and phasic increase in proinflammatory cytokines, reduces migraine frequency. Thus, we applied phasic or pulsed IFNγ to brain slice cultures to emulate EE. This treatment reduced OS, increased myelin basic protein, a marker for myelin, and reduced susceptibility to SD. Building on our research on exosomes in EE-based neuroprotection, we found that IFNγ stimulation of slice cultures induced release of exosomes, likely from the microglia that produce the same protective effects as IFNγ treatment when applied to naive cultures. Finally, nasal administration of IFNγ to rats recapitulated in vitro effects, reducing OS, increasing myelin, and reducing SD. These results support phasic IFNγ signaling as a therapeutic target for prevention of SD and, by extension, migraine.

Spreading depression transiently disrupts myelin via interferon-gamma signaling.

Multiple sclerosis and migraine with aura are clinically correlated and both show imaging changes suggestive of myelin disruption. Furthermore, cortical myelin loss in the cuprizone animal model of multiple sclerosis enhances susceptibility to spreading depression, the likely underlying cause of migraine with aura. Since multiple sclerosis pathology involves inflammatory T cell lymphocyte production of interferon-gamma and a resulting increase in oxidative stress, we tested the hypothesis that spreading depression disrupts myelin through similar signaling pathways. Rat hippocampal slice cultures were initially used to explore myelin loss in spreading depression, since they contain T cells, and allow for controlled tissue microenvironment. These experiments were then translated to the in vivo condition in neocortex. Spreading depression in slice cultures induced significant loss of myelin integrity and myelin basic protein one day later, with gradual recovery by seven days. Myelin basic protein loss was abrogated by T cell depletion, neutralization of interferon-gamma, and pharmacological inhibition of neutral sphingomyelinase-2. Conversely, one day after exposure to interferon-gamma, significant reductions in spreading depression threshold, increases in oxidative stress, and reduced levels of glutathione, an endogenous neutral sphingomyelinase-2 inhibitor, emerged. Similarly, spreading depression triggered significant T cell accumulation, sphingomyelinase activation, increased oxidative stress, and reduction of gray and white matter myelin in vivo. Myelin disruption is involved in spreading depression, thereby providing pathophysiological links between multiple sclerosis and migraine with aura. Myelin disruption may promote spreading depression by enhancing aberrant excitability. Thus, preservation of myelin integrity may provide novel therapeutic targets for migraine with aura.

Spreading depression requires microglia and is decreased by their M2a polarization from environmental enrichment.

Microglia play an important role in fine-tuning neuronal activity. In part, this involves their production of tumor necrosis factor-alpha (TNFα), which increases neuronal excitability. Excessive synaptic activity is necessary to initiate spreading depression (SD). Increased microglial production of proinflammatory cytokines promotes initiation of SD, which, when recurrent, may play a role in conversion of episodic to high frequency and chronic migraine. Previous work shows that this potentiation of SD occurs through increased microglial production of TNFα and reactive oxygen species, both of which are associated with an M1-skewed microglial population. Hence, we explored the role of microglia and their M1 polarization in SD initiation. Selective ablation of microglia from rat hippocampal slice cultures confirmed that microglia are essential for initiation of SD. Application of minocycline to dampen M1 signaling led to increased SD threshold. In addition, we found that SD threshold was increased in rats exposed to environmental enrichment. These rats had increased neocortical levels of interleukin-11 (IL-11), which decreases TNFα signaling and polarized microglia to an M2a-dominant phenotype. M2a microglia reduce proinflammatory signaling and increase production of anti-inflammatory cytokines, and therefore may protect against SD. Nasal administration of IL-11 to mimic effects of environmental enrichment likewise increased M2a polarization and increased SD threshold, an effect also seen in vitro. Similarly, application of conditioned medium from M2a polarized primary microglia to slice cultures also increased SD threshold. Thus, microglia and their polarization state play an essential role in SD initiation, and perhaps by extension migraine with aura and migraine.

What are exosomes and how can they be used in multiple sclerosis therapy?

Current treatment options for multiple sclerosis are limited and consist of immunosuppressors or agents to prevent immune infiltration of the brain. These therapies have potentially harmful side effects and do little to promote myelin repair. Instead, we suggest using exosomes, naturally occurring small vesicles that exert influence through the delivery of mRNA, microRNA and protein. Dendritic cells can be cultured from bone marrow and stimulated to release exosomes. When administered to the brain, these exosomes significantly increase myelination and improve remyelination following injury by prompting preoligodendrocytes to differentiate into myelin producing cells. Additionally, they are non-toxic and can easily cross the blood-brain barrier and, thus, have great potential as a therapeutic.

IFNγ-stimulated dendritic cell exosomes as a potential therapeutic for remyelination.

Dendritic cells (DCs) release exosomes with different characteristics based on stimulus. Here, we showed that DC cultures stimulated with low-level IFNγ released exosomes (IFNγ-DC-Exos) that contained microRNA species that can increase baseline myelination, reduce oxidative stress, and improve remyelination following acute lysolecithin-induced demyelination. Furthermore, nasally administered IFNγ-DC-Exos increased CNS myelination in vivo. IFNγ-DC-Exos were preferentially taken up by oligodendrocytes, suggesting that they directly impact oligodendrocytes to increase myelination. Thus, our results show great potential for use of these IFNγ-DC-Exos as a therapeutic to promote remyelination in multiple sclerosis and dysmyelinating syndromes.

Youth and environmental enrichment generate serum exosomes containing miR-219 that promote CNS myelination.

Although commonly considered a disease of white matter, gray matter demyelination is increasingly recognized as an important component of multiple sclerosis (MS) pathogenesis, particularly in the secondary progressive disease phase. Extent of damage to gray matter is strongly correlated to decline in memory and cognitive dysfunction in MS patients. Aging likewise occurs with cognitive decline from myelin loss, and age-associated failure to remyelinate significantly contributes to MS progression. However, recent evidence demonstrates that parabiotic exposure of aged animals to a youthful systemic milieu can promote oligodendrocyte precursor cell (OPC) differentiation and improve remyelination. In the current study, we focus on this potential for stimulating remyelination, and show it involves serum exosomes that increase OPCs and their differentiation into mature myelin-producing cells-both under control conditions and after acute demyelination. Environmental enrichment (EE) of aging animals produced exosomes that mimicked this promyelinating effect. Additionally, stimulating OPC differentiation via exosomes derived from environmentally enriched animals is unlikely to deplete progenitors, as EE itself promotes proliferation of neural stem cells. We found that both young and EE serum-derived exosomes were enriched in miR-219, which is necessary and sufficient for production of myelinating oligodendrocytes by reducing the expression of inhibitory regulators of differentiation. Accordingly, protein transcript levels of these miR-219 target mRNAs decreased following exosome application to slice cultures. Finally, nasal administration of exosomes to aging rats also enhanced myelination. Thus, peripheral circulating cells in young or environmentally enriched animals produce exosomes that may be a useful therapy for remyelination.

Insulin-like growth factor-1 abrogates microglial oxidative stress and TNF-α responses to spreading depression.

Spreading depression (SD), the most likely cause of migraine aura and perhaps migraine, occurs with increased oxidative stress (OS). SD increases reactive oxygen species (ROS), and ROS, in turn, can signal to increase neuronal excitability,which includes increased SD susceptibility. SD also elevates tumor necrosis factor-α (TNF-α), which increases neuronal excitability. Accordingly, we probed for the cellular origin of OS from SD and its relationship to TNF-α, which might promote SD, using rat hippocampal slice cultures. We observed significantly increased OS from SD in astrocytes and microglia but not in neurons or oligodendrocytes. Since insulin-like growth factor-1 (IGF-1) mitigates OS from SD, we determined the cell types responsible for this effect. We found that IGF-1 significantly decreased microglial but not astrocytic OS from SD. We also show that IGF-1 abrogated the SD-induced TNF-α increase. Furthermore, TNF-α application increased microglial but not astrocytic OS, an effect abrogated by IGF-1. Next,we showed that SD increased SD susceptibility, and does so via TNF-α. This work suggests that microglia promote SD via increased and interrelated ROS and TNF-α signaling. Thus, IGF-1 mitigation of microglial ROS and TNF-α responses maybe targets for novel therapeutics development to prevent SD, and perhaps migraine.

Nanoparticle targeting to neurons in a rat hippocampal slice culture model.

We have previously shown that CdSe/ZnS core/shell luminescent semiconductor nanocrystals or QDs (quantum dots) coated with PEG [poly(ethylene glycol)]-appended DHLA (dihydrolipoic acid) can bind AcWG(Pal)VKIKKP(9)GGH(6) (Palm1) through the histidine residues. The coating on the QD provides colloidal stability and this peptide complex uniquely allows the QDs to be taken up by cultured cells and readily exit the endosome into the soma. We now show that use of a polyampholyte coating [in which the neutral PEG is replaced by the negatively heterocharged CL4 (compact ligand)], results in the specific targeting of the palmitoylated peptide to neurons in mature rat hippocampal slice cultures. There was no noticeable uptake by astrocytes, oligodendrocytes or microglia (identified by immunocytochemistry), demonstrating neuronal specificity to the overall negatively charged CL4 coating. In addition, EM (electron microscopy) images confirm the endosomal egress ability of the Palm1 peptide by showing a much more disperse cytosolic distribution of the CL4 QDs conjugated to Palm1 compared with CL4 QDs alone. This suggests a novel and robust way of delivering neurotherapeutics to neurons.

Insulin-like growth factor-1 lowers spreading depression susceptibility and reduces oxidative stress.

Spreading depression (SD), the likely cause of migraine aura and perhaps migraine, is triggered by widespread and unfettered neuronal hyperexcitability. Migraine and the initiating hyperexcitability of seizure, which involve oxidative stress (OS), are likely interrelated. Environmental enrichment (EE) decreases seizure and can reduce migraine. EE's well-characterized neuroprotective effect involves insulin-like growth factor-1 (IGF-1). Accordingly, we asked if IGF-1 could mitigate the hyperexcitability that initiates SD using rat hippocampal slice cultures. We demonstrate that IGF-1 significantly decreased SD susceptibility and related OS. We mimicked OS of SD and observed that IGF-1 abolished hyperexcitability from OS. Application of an antioxidant significantly decreased SD susceptibility and co-administration of an antioxidant with IGF-1 produced no additive effect, whereas an oxidizer significantly increased SD, and this effect was abrogated by IGF-1. Moreover, IGF-1 significantly decreased baseline OS, despite seemingly paradoxically increasing CA3 bursting. These results suggest that IGF-1 increased endogenous antioxidants to levels sufficient to buffer against the OS of SD. Insulin similarly mitigated SD susceptibility, but required a far greater dose. Since brain IGF-1 increases with EE, and, like insulin, independently functions as an EE mimetic, we suggest that EE mimetics are a novel source of therapeutics for SD, and by extension, migraine.

Pregnant serum induces neuroinflammation and seizure activity via TNFα.

Preeclampsia is a hypertensive disorder of pregnancy that affects many organs including the brain. Neurological complications occur during preeclampsia, the most serious of which is seizure known as eclampsia. Although preeclampsia can precede the eclamptic seizure, it often occurs during normal pregnancy, suggesting that processes associated with normal pregnancy can promote neuronal excitability. Here we investigated whether circulating inflammatory mediators that are elevated late in gestation when seizure also occurs are hyperexcitable to neuronal tissue. Evoked field potentials were measured in hippocampal slices in which control horse serum that slices are normally grown in, was replaced with serum from nonpregnant or late-pregnant Wistar rats for 48 h. We found that serum from pregnant, but not nonpregnant rats, caused hyperexcitability to hippocampal neurons and seizure activity that was abrogated by inhibition of tumor necrosis factor alpha (TNFα) signaling. Additionally, application of TNFα mimicked this increased excitability. Pregnant serum also caused morphological changes in microglia characteristic of activation, and increased TNFα mRNA expression that was not seen with exposure to nonpregnant serum. However, TNFα protein was not found to be elevated in pregnant serum itself, suggesting that other circulating factors during pregnancy caused activation of hippocampal slice cells to produce a TNFα-mediated increase in neuronal excitability. Lastly, although pregnant serum caused neuroinflammation and hyperexcitability of hippocampal slices, it did not increase blood-brain barrier permeability, nor were pregnant rats from which the serum was taken undergoing seizure. Thus, the BBB has an important role in protecting the brain from circulating neuroinflammatory mediators that are hyperexcitable to the brain during pregnancy. These studies provide novel insight into the underlying cause of eclampsia without elevated blood pressure and the protective role of the BBB that prevents exposure of the brain to hyperexcitable factors.

Differential effects of low-dose endotoxin on the cerebral circulation during pregnancy.

It is well-known that the pregnant state is associated with increased sensitivity to endotoxin in renal and uterine circulations; however, the effects on the cerebral circulation are not known. Intravenous infusion of low-dose lipopolysaccharide ([LPS]; 1.5 µg/kg) to pregnant Wistar rats on day 15 of pregnancy caused significantly decreased myogenic tone of posterior cerebral arteries on day 20, which was not seen in similarly treated nonpregnant rats. Pregnancy alone was associated with a 2-to 4-fold increase in inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) messenger RNA (mRNA) in cerebral arteries compared to nonpregnant, suggesting that the cerebral circulation is in a state of inflammation during pregnancy. After LPS treatment, cerebral arteries from pregnant animals had increased iNOS and TNF-α compared to LPS-treated nonpregnant animals, but decreased interleukin 10 (IL-10) and IFN-γ. These results demonstrate that pregnancy enhances sensitivity to the effects of LPS in the cerebral circulation, which may be due to an enhanced inflammatory state during pregnancy.

Modeling neural immune signaling of episodic and chronic migraine using spreading depression in vitro.

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.