In vitro biocompatibility evaluation of functional electrically stimulating microelectrodes on primary glia.

Neural interfacing devices interact with the central nervous system to alleviate functional deficits arising from disease or injury. This often entails the use of invasive microelectrode implants that elicit inflammatory responses from glial cells and leads to loss of device function. Previous work focused on improving implant biocompatibility by modifying electrode composition; here, we investigated the direct effects of electrical stimulation on glial cells at the electrode interface. A high-throughput in vitro system that assesses primary glial cell response to biphasic stimulation waveforms at 0 mA, 0.15 mA, and 1.5 mA was developed and optimized. Primary mixed glial cell cultures were generated from heterozygous CX3CR-1+/EGFP mice, electrically stimulated for 4 h/day over 3 days using 75 µm platinum-iridium microelectrodes, and biomarker immunofluorescence was measured. Electrodes were then imaged on a scanning electron microscope to assess sustained electrode damage. Fluorescence and electron microscopy analyses suggest varying degrees of localized responses for each biomarker assayed (Hoescht, EGFP, GFAP, and IL-1ß), a result that expands on comparable in vivo models. This system allows for the comparison of a breadth of electrical stimulation parameters, and opens another avenue through which neural interfacing device developers can improve biocompatibility and longevity of electrodes in tissue.

Short-chain fatty and carboxylic acid changes associated with fecal microbiota transplant communally influence microglial inflammation.

The intestinal microbiota has been proposed to influence human mental health and cognition through the gut-brain axis. Individuals experiencing recurrent Clostridioides difficile infection (rCDI) frequently report depressive symptoms, which are improved after fecal microbiota transplantation (FMT); however, mechanisms underlying this association are poorly understood. Short-chain fatty acids and carboxylic acids (SCCA) produced by the intestinal microbiota cross the blood brain barrier and have been proposed to contribute to gut-brain communication. We hypothesized that changes in serum SCCA measured before and after successful FMT for rCDI influences the inflammatory response of microglia, the resident immune cells of the central nervous system. Serum SCCA were quantified using gas chromatography-mass spectroscopy from 38 patients who participated in a randomized trial comparing oral capsule-vs colonoscopy-delivered FMT for rCDI, and quality of life was assessed by SF-36 at baseline, 4, and 12 weeks after FMT treatment. Successful FMT was associated with improvements in mental and physical health, as well as significant changes in a number of circulating SCCA, including increased butyrate, 2-methylbutyrate, valerate, and isovalerate, and decreased 2-hydroxybutyrate. Primary cultured microglia were treated with SCCA and the response to a pro-inflammatory stimulus was measured. Treatment with a combination of SCCA based on the post-FMT serum profile, but not single SCCA species, resulted in significantly reduced inflammatory response including reduced cytokine release, reduced nitric oxide release, and accumulation of intracellular lipid droplets. This suggests that both levels and diversity of SCCA may be an important contributor to gut-brain communication.

Pleiotrophin Signals Through ALK Receptor to Enhance the Growth of Neurons in the Presence of Inhibitory Chondroitin Sulfate Proteoglycans.

Chondroitin sulfate proteoglycans (CSPGs), one of the major extracellular matrix components of the glial scar that surrounds central nervous system (CNS) injuries, are known to inhibit the regeneration of neurons. This study investigated whether pleiotrophin (PTN), a growth factor upregulated during early CNS development, can overcome the inhibition mediated by CSPGs and promote the neurite outgrowth of neurons in vitro. The data showed that a CSPG matrix inhibited the outgrowth of neurites in primary cortical neuron cultures compared to a control matrix. PTN elicited a dose-dependent increase in the neurite outgrowth even in the presence of the growth inhibitory CSPG matrix, with optimal growth at 15 ng mL-1 of PTN (114.8% of neuronal outgrowth relative to laminin control). The growth-promoting effect of PTN was blocked by inhibition of the receptor anaplastic lymphoma kinase (ALK) by alectinib in a dose-dependent manner. Neurite outgrowth in the presence of this CSPG matrix was induced by activation of the protein kinase B (AKT) pathway, a key downstream mediator of ALK activation. This study identified PTN as a dose-dependent regulator of neurite outgrowth in primary cortical neurons cultured in the presence of a CSPG matrix and identified ALK activation as a key driver of PTN-induced growth.

The effects of electrical stimulation on glial cell behaviour.

Neural interface devices interact with the central nervous system (CNS) to substitute for some sort of functional deficit and improve quality of life for persons with disabilities. Design of safe, biocompatible neural interface devices is a fast-emerging field of neuroscience research. Development of invasive implant materials designed to directly interface with brain or spinal cord tissue has focussed on mitigation of glial scar reactivity toward the implant itself, but little exists in the literature that directly documents the effects of electrical stimulation on glial cells. In this review, a survey of studies documenting such effects has been compiled and categorized based on the various types of stimulation paradigms used and their observed effects on glia. A hybrid neuroscience cell biology-engineering perspective is offered to highlight considerations that must be made in both disciplines in the development of a safe implant. To advance knowledge on how electrical stimulation affects glia, we also suggest experiments elucidating electrochemical reactions that may occur as a result of electrical stimulation and how such reactions may affect glia. Designing a biocompatible stimulation paradigm should be a forefront consideration in the development of a device with improved safety and longevity.

Overview of the Neuroprotective Effects of the MAO-Inhibiting Antidepressant Phenelzine.

Phenelzine (PLZ) is a monoamine oxidase (MAO)-inhibiting antidepressant with anxiolytic properties. This multifaceted drug has a number of pharmacological and neurochemical effects in addition to inhibition of MAO, and findings on these effects have contributed to a body of evidence indicating that PLZ also has neuroprotective/neurorescue properties. These attributes are reviewed in this paper and include catabolism to the active metabolite ß-phenylethylidenehydrazine (PEH) and effects of PLZ and PEH on the GABA-glutamate balance in brain, sequestration of reactive aldehydes, and inhibition of primary amine oxidase. Also discussed are the encouraging findings of the effects of PLZ in animal models of stroke, spinal cord injury, traumatic brain injury, and multiple sclerosis, as well other actions such as reduction of nitrative stress, reduction of the effects of a toxin on dopaminergic neurons, potential anticonvulsant actions, and effects on brain-derived neurotrophic factor, neural cell adhesion molecules, an anti-apoptotic factor, and brain levels of ornithine and N-acetylamino acids.

Inflammatory Cytokine Profile and Plasticity of Brain and Spinal Microglia in Response to ATP and Glutamate.

Microglia are the primary cells in the central nervous system that identify and respond to injury or damage. Such a perturbation in the nervous system induces the release of molecules including ATP and glutamate that act as damage-associated molecular patterns (DAMPs). DAMPs are detected by microglia, which then regulate the inflammatory response in a manner sensitive to their surrounding environment. The available data indicates that ATP and glutamate can induce the release of pro inflammatory factors TNF (tumor necrosis factor), IL-1ß (interleukin 1 beta), and NO (nitric oxide) from microglia. However, non-physiological concentrations of ATP and glutamate were often used to derive these insights. Here, we have compared the response of spinal cord microglia (SM) relative to brain microglia (BM) using physiologically relevant concentrations of glutamate and ATP that mimic injured conditions in the central nervous system. The data show that ATP and glutamate are not significant modulators of the release of cytokines from either BM or SM. Consistent with previous studies, spinal microglia exhibited a general trend toward reduced release of inflammatory cytokines relative to brain-derived microglia. Moreover, we demonstrate that the responses of microglia to these DAMPs can be altered by modifying the biochemical milieu in their surrounding environment. Preconditioning brain derived microglia with media from spinal cord derived mixed glial cultures shifted their release of IL-1ß and IL-6 to a less inflammatory phenotype consistent with spinal microglia.

Applying a novel 3D hydrogel cell culture to investigate activation of microglia due to rotational kinematics associated with mild traumatic brain injury.

Many investigations on mild traumatic brain injury (mTBI) aim to further understand how cells in the brain react to the mechanical forces associated with the injury. While it is known that rapid head rotation is a mechanism contributing to mTBI, establishing definitive thresholds for head rotation has proved challenging. One way to advance determining mechanisms and thresholds for injury is through in vitro models. Here, an apparatus has been designed that is capable of delivering rotational forces to three-dimensional (3D) hydrogel cell cultures. Using an in vitro model, we test the hypothesis that rotational kinematics can activate microglia suspended in a 3-dimensional mixed glia environment (absent neurons). The impact apparatus was able to deliver peak angular velocities of approximately 45 rad/s, a magnitude for angular velocity that in select literature is associated with diffuse brain injury. However, no measurable glial cell reactivity was observed in response to the rotational kinematics through any of the chosen metrics (nitric oxide, pro-inflammatory cytokine release and proportion of amoeboid activated microglia). The results generated from this study suggest that rotation of the glia alone did not cause activation - in future work we will investigate the effect of neuronal contributions in activating glia.

In Vitro Priming and Hyper-Activation of Brain Microglia: an Assessment of Phenotypes.

Microglia are the resident immune cells of the central nervous system that mediate the life and death of nervous tissue. During normal function, they exhibit a surveying phenotype and maintain vital functions in nervous tissue. In the event of injury or disease, chronic inflammation can result, wherein microglia develop a hyper-activated phenotype, shed their regenerative function, actively kill contiguous cells, and can partition injured tissue by initiating scar formation. With recoverable injury, microglia can develop a primed phenotype, where they appear to recover from an inflammatory event, but are limited in their support functions and show inappropriate responses to future injury often associated with neurodegenerative disorders. These microglial phenotypes were acutely recreated in vitro with potent pro- and anti-inflammatory treatments. Primary cultured microglia or mixed glia (microglia, astrocytes, and oligodendrocytes) were treated for 6 h with lipopolysaccharide (LPS). Recovery from an inflammatory state was modeled with 18-h treatment of the anti-inflammatory steroid dexamethasone. The cells were then treated for 24 h with interferon gamma (IFNγ) to detect inflammatory memory after recovery. Surveying was best represented in the untreated vehicle (Veh) cases and was characterized by negligible secretion of pro-inflammatory factors, limited expression of immune proteins such as induced nitric oxide synthase (iNOS), major histocompatibility complex class II (MHCII), relatively high expression of brain-derived and glial-derived neurotrophic factors (BDNF and GDNF), and thinly branched smaller microglia. Activation was noted in the LPS- and IFNγ-treated microglia with increased cytokines, NO, NGF, iNOS, proliferation, phagocytosis, reduced BDNF, and flattened round amoeboid-shaped microglia. Priming was observed to be an incomplete surveying restoration using dexamethasone from an activation comparison of LPS, IFNγ, and LPS/IFNγ. Dexamethasone treatments resulted in the most profound dysregulation of expression of NO, TNF, IL-1ß, NGF, CD68, and MHCII as well as ramified morphology and uptake of myelin. These findings suggest microglial priming and hyper-activation may be effectively modeled in vitro to allow mechanistic investigations into these key cellular phenotypes.

Fluoxetine and its metabolite norfluoxetine induce microglial apoptosis.

Inflammatory insult to the central nervous system (CNS) can lead to development of depression, and subsequently depression is the most frequent psychiatric comorbidity following ischemic stroke, often limiting recovery and rehabilitation in patients. The initiators of inflammatory pathways in the CNS are microglia activated in response to acute ischemic stress, and anti-depressants have been shown to have anti-inflammatory effects in the CNS, promoting neuronal survival following ischemic insult. We have previously shown that the selective serotonin reuptake inhibitors (SSRIs) fluoxetine and citalopram promote neuronal survival after oxygen-glucose deprivation, an in vitro model of ischemia, by attenuating the release of glutamate and D-serine from activated microglia. Interestingly, we found that fluoxetine-treated microglial cultures contained fewer numbers of cells compared to other groups and hypothesized that fluoxetine and citalopram attenuated the release of glutamate and D-serine by promoting the apoptosis of microglia. The present study aimed to test and compare antidepressants from three distinct classes (tricyclics, monoamine oxidase inhibitors, and SSRIs) on microglial apoptosis. Primary microglia were treated with 1 µg/mL lipopolysaccharide and/or 10 µM antidepressants, and various apoptotic markers were assayed. Fluoxetine and its metabolite norfluoxetine decreased protein levels in cell lysates, decreased cell viability of microglia, and increased the expression of the apoptotic marker cleaved-caspase 3 in microglia. Live/dead nuclear staining also showed that fluoxetine- or norfluoxetine-treated cultures contained greater numbers of dying microglial cells compared to vehicle-treated cultures. Cultures treated with citalopram, phenelzine, or imipramine showed no evidence of inducing microglial apoptosis. Our results demonstrate that fluoxetine and norfluoxetine induce the apoptotic death of microglia, which may serve as a mechanism to attenuate the release of glutamate and D-serine from activated microglia. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Microglial Function during Glucose Deprivation: Inflammatory and Neuropsychiatric Implications.

Inflammation is increasingly recognized as a contributor to the pathophysiology of neuropsychiatric disorders, including depression, anxiety disorders and autism, though the factors leading to contextually inappropriate or sustained inflammation in pathological conditions are yet to be elucidated. Microglia, as the key mediators of inflammation in the CNS, serve as likely candidates in initiating pathological inflammation and as an ideal point of therapeutic intervention. Glucose deprivation, as a component of the pathophysiology of ischemia or occurring transiently in diabetes, may serve to modify microglial function contributing to inflammatory injury. To this end, primary microglia were cultured from postnatal rat brain and subject to glucose deprivation in vitro. Microglia were characterized for their proliferation, phagocytic function and secretion of inflammatory factors, and tested for their capacity to respond to a potent inflammatory stimulus. In the absence of glucose, microglia remained capable of proliferation, phagocytosis and inflammatory activation and showed increased release of inflammatory factors after presentation of an inflammatory stimulus. Glucose-deprived microglia demonstrated increased phagocytic activity and decreased accumulation of lipids in lipid droplets over a 48-h timecourse, suggesting they may use scavenged lipids as a key alternate energy source during metabolic stress. In the present manuscript, we present novel findings that glucose deprivation may sensitize microglial release of inflammatory mediators and prime microglial functions for both survival and inflammatory roles, which may contribute to psychiatric comorbidities of ischemia, diabetes and/or metabolic disorder.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation.

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Disease-modifying effects of ganglioside GM1 in Huntington's disease models.

Huntington's disease (HD) is a progressive neurodegenerative disorder characterized by motor, cognitive and psychiatric problems. Previous studies indicated that levels of brain gangliosides are lower than normal in HD models and that administration of exogenous ganglioside GM1 corrects motor dysfunction in the YAC128 mouse model of HD In this study, we provide evidence that intraventricular administration of GM1 has profound disease-modifying effects across HD mouse models with different genetic background. GM1 administration results in decreased levels of mutant huntingtin, the protein that causes HD, and in a wide array of beneficial effects that include changes in levels of DARPP32, ferritin, Iba1 and GFAP, modulation of dopamine and serotonin metabolism, and restoration of normal levels of glutamate, GABA, L-Ser and D-Ser. Treatment with GM1 slows down neurodegeneration, white matter atrophy and body weight loss in R6/2 mice. Motor functions are significantly improved in R6/2 mice and restored to normal in Q140 mice, including gait abnormalities that are often resistant to treatments. Psychiatric-like and cognitive dysfunctions are also ameliorated by GM1 administration in Q140 and YAC128 mice. The widespread benefits of GM1 administration, at molecular, cellular and behavioural levels, indicate that this ganglioside has strong therapeutic and disease-modifying potential in HD.

Peptide modification of polyimide-insulated microwires: Towards improved biocompatibility through reduced glial scarring.

The goal of this study is to improve the integration of implanted microdevices with tissue in the central nervous system (CNS). The long-term utility of neuroprosthetic devices implanted in the CNS is affected by the formation of a scar by resident glial cells (astrocytes and microglia), limiting the viability and functional stability of the devices. Reduction in the proliferation of glial cells is expected to enhance the biocompatibility of devices. We demonstrate the modification of polyimide-insulated microelectrodes with a bioactive peptide KHIFSDDSSE. Microelectrode wires were functionalized with (3-aminopropyl) triethoxy silane (APTES); the peptide was then covalently bonded to the APTES. The soluble peptide was tested in 2D mixed cultures of astrocytes and microglia, and reduced the proliferation of both cell types. The interactions of glial cells with the peptide-modified wires was then examined in 3D cell-laden hydrogels by immunofluorescence microscopy. As expected for uncoated wires, the microglia were first attracted to the wire (7days) followed by astrocyte recruitment and hypertrophy (14days). For the peptide-treated wires, astrocytes coated the wires directly (24h), and formed a thin, stable coating without evidence of hypertrophy, and the attraction of microglia to the wire was significantly reduced. The results suggest a mechanism to improve tissue integration by promoting uniform coating of astrocytes on a foreign body while lessening the reactive response of microglia. We conclude that the bioactive peptide KHIFSDDSSE may be effective in improving the biocompatibility of neural interfaces by both reducing acute glial reactivity and generating stable integration with tissue. STATEMENT OF SIGNIFICANCE: The peptide KHIFSDDSSE has previously been shown in vitro to both reduce the proliferation of astrocytes, and to increase the adhesion of astrocyte to glass substrates. Here, we demonstrate a method to apply uniform coatings of peptides to microwires, which could readily be generalized to other peptides and surfaces. We then show that when peptide-modified wires are inserted into 3D cell-laden hydrogels, the normal cellular reaction (microglial activation followed by astrocyte recruitment and hypertrophy) does not occur, rather astrocytes are attracted directly to the surface of the wire, forming a relatively thin and uniform coating. This suggests a method to improve tissue integration of implanted devices to reduce glial scarring and ultimately reduce failure of neural interfaces.

Transient Aortic Occlusion Augments Collateral Blood Flow and Reduces Mortality During Severe Ischemia due to Proximal Middle Cerebral Artery Occlusion.

Cerebral collateral circulation provides alternative vascular routes for blood to reach ischemic tissues during stroke. Collateral therapeutics attempt to augment flow through these collateral channels to reduce ischemia and brain damage during acute ischemic stroke. Transient aortic occlusion (TAO) has pre-clinical data suggesting that it can augment collateral blood flow and clinical data suggesting a benefit for patients with moderate cortical strokes. By diverting blood from the periphery towards the cerebral circulation, TAO has the potential to augment primary collateral flow at the circle of Willis and thereby improve outcome even during large, hemispheric strokes. Using proximal middle and anterior cerebral artery occlusion in rats, we demonstrate that TAO reduces mortality and improves collateral blood flow in severely ischemic animals. As such, TAO may be an effective therapy to reduce early mortality during severe ischemia associated with proximal occlusions.

Statin treatment affects cytokine release and phagocytic activity in primary cultured microglia through two separable mechanisms.

BACKGROUND: As the primary immune cells of the central nervous system, microglia contribute to development, homeostasis, and plasticity of the central nervous system, in addition to their well characterized roles in the foreign body and inflammatory responses. Increasingly, inappropriate activation of microglia is being reported as a component of inflammation in neurodegenerative and neuropsychiatric disorders. The statin class of cholesterol-lowering drugs have been observed to have anti-inflammatory and protective effects in both neurodegenerative diseases and ischemic stroke, and are suggested to act by attenuating microglial activity. RESULTS: We sought to investigate the effects of simvastatin treatment on the secretory profile and phagocytic activity of primary cultured rat microglia, and to dissect the mechanism of action of simvastatin on microglial activity. Simvastatin treatment altered the release of cytokines and trophic factors from microglia, including interleukin-1-ß, tumour necrosis factor-α, and brain derived neurotrophic factor in a cholesterol-dependent manner. Conversely, simvastatin inhibited phagocytosis in microglia in a cholesterol-independent manner. CONCLUSIONS: The disparity in cholesterol dependence of cytokine release and phagocytosis suggests the two effects occur through distinct molecular mechanisms. These two pathways may provide an opportunity for further refinement of pharmacotherapies for neuroinflammatory, neurodegenerative, and neuropsychiatric disorders.

Reduced inflammatory phenotype in microglia derived from neonatal rat spinal cord versus brain.

Microglia are the primary immune cells of the central nervous system (CNS). Membrane bound sensors on their processes monitor the extracellular environment and respond to perturbations of the CNS such as injury or infection. Once activated, microglia play a crucial role in determining neuronal survival. Recent studies suggest that microglial functional response properties vary across different regions of the CNS. However, the activation profiles of microglia derived from the spinal cord have not been evaluated against brain microglia in vitro. Here, we studied the morphological properties and secretion of inflammatory and trophic effectors by microglia derived from the brain or spinal cord of neonatal rats under basal culture conditions and after activation with lipopolysaccharide (LPS). Our results demonstrate that spinal microglia assume a less inflammatory phenotype after LPS activation, with reduced release of the inflammatory effectors tumor necrosis factor alpha, interleukin-1 beta, and nitric oxide, a less amoeboid morphology, and reduced phagocytosis relative to brain-derived microglia. Phenotypic differences between brain and spinal microglia are an important consideration when evaluating anti-inflammatory or immunomodulatory therapies for brain versus spinal injury.

Hyaluronic acid-based 3D culture model for in vitro testing of electrode biocompatibility.

This work describes the development of a robust and repeatable in vitro 3D culture model of glial scarring, which may be used to evaluate the foreign body response to electrodes and other implants in the central nervous system. The model is based on methacrylated hyaluronic acid, a hydrogel that may be photopolymerized to form an insoluble network. Hydrogel scaffolds were formed at four different macromer concentrations (0.50, 0.75, 1.00, and 1.50% (w/v)). As expected, the elastic modulus of the scaffolds increased with increasing macromer weight fraction. Adult rat brain tested under identical conditions had an elastic modulus range that spanned the elastic modulus of both the 0.50 and 0.75% (w/v) hydrogel samples. Gels formed with higher macromer weight fraction had decreased equilibrium swelling ratio and visibly thicker pore walls relative to gels formed with lower macromer weight fractions. Mixed glial cells (microglia and astrocytes) were then encapsulated in the HA scaffolds. Viability of the mixed cultures was most stable at a cell density of 1 × 10(7) cells/mL. Cell viability at the highest macromer weight fraction tested (1.50% (w/v)) was significantly lower than other tested gels (0.50, 0.75 and 1.00% (w/v)). The inflammatory response of microglia and astrocytes to a microelectrode inserted into the scaffold was assessed over a period of 2 weeks and closely represented that reported in vivo. Microglia responded first to the electrode (increased cell density at the electrode, and activated morphology) followed by astrocytes (appeared to line the electrode in a manner similar to glial scarring). All together, these results demonstrate the potential of the 3D in vitro model system to assess glial scarring in a robust and repeatable manner.

Augmenting collateral blood flow during ischemic stroke via transient aortic occlusion.

Collateral circulation provides an alternative route for blood flow to reach ischemic tissue during a stroke. Blood flow through the cerebral collaterals is a critical predictor of clinical prognosis after stroke and response to recanalization, but data on collateral dynamics and collateral therapeutics are lacking. Here, we investigate the efficacy of a novel approach to collateral blood flow augmentation to increase collateral circulation by optically recording blood flow in leptomeningeal collaterals in a clinically relevant model of ischemic stroke. Using high-resolution laser speckle contrast imaging (LSCI) during thromboembolic middle cerebral artery occlusion (MCAo), we demonstrate that transiently diverting blood flow from peripheral circulation towards the brain via intra-aortic catheter and balloon induces persistent increases in blood flow through anastomoses between the anterior and middle cerebral arteries. Increased collateral flow restores blood flow in the distal middle cerebral artery segments to baseline levels during aortic occlusion and persists for over 1 hour after removal of the aortic balloon. Given the importance of collateral circulation in predicting stroke outcome and response to treatment, and the potential of collateral flow augmentation as an adjuvant or stand-alone therapy for acute ischemic stroke, this data provide support for further development and translation of collateral therapeutics including transient aortic occlusion.

Antenatal sildenafil treatment attenuates pulmonary hypertension in experimental congenital diaphragmatic hernia.

BACKGROUND: Lung hypoplasia and persistent pulmonary hypertension of the newborn limit survival in congenital diaphragmatic hernia (CDH). Unlike other diseases resulting in persistent pulmonary hypertension of the newborn, infants with CDH are refractory to inhaled nitric oxide (NO). Nitric oxide mediates pulmonary vasodilatation at birth in part via cyclic GMP production. Phosphodiesterase type 5 (PDE5) limits the effects of NO by inactivation of cyclic GMP. Because of the limited success in postnatal management of CDH, we hypothesized that antenatal PDE5 inhibition would attenuate pulmonary artery remodeling in experimental nitrofen-induced CDH. METHODS AND RESULTS: Nitrofen administered at embryonic day 9.5 to pregnant rats resulted in a 60% incidence of CDH in the offspring and recapitulated features seen in human CDH, including structural abnormalities (lung hypoplasia, decreased pulmonary vascular density, pulmonary artery remodeling, right ventricular hypertrophy), and functional abnormalities (decreased pulmonary artery relaxation in response to the NO donor 2-(N,N-diethylamino)-diazenolate-2-oxide). Antenatal sildenafil administered to the pregnant rat from embryonic day 11.5 to embryonic day 20.5 crossed the placenta, increased fetal lung cyclic GMP and decreased active PDE5 expression. Antenatal sildenafil improved lung structure, increased pulmonary vessel density, reduced right ventricular hypertrophy, and improved postnatal NO donor 2-(N,N-diethylamino)-diazenolate-2-oxide-induced pulmonary artery relaxation. This was associated with increased lung endothelial NO synthase and vascular endothelial growth factor protein expression. Antenatal sildenafil had no adverse effect on retinal structure/function and brain development. CONCLUSIONS: Antenatal sildenafil improves pathological features of persistent pulmonary hypertension of the newborn in experimental CDH and does not alter the development of other PDE5-expressing organs. Given the high mortality/morbidity of CDH, the potential benefit of prenatal PDE5 inhibition in improving the outcome for infants with CDH warrants further studies.