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.

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/.

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.

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.

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.

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.

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.

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.

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.

The antidepressant phenelzine protects neurons and astrocytes against formaldehyde-induced toxicity.

Reactive aldehydes have been implicated in the etiology of several neurological and psychiatric disorders, and there is considerable interest in drugs to counteract the actions of these aldehydes. Increased formaldehyde (FA) and up-regulation of semicarbazide-sensitive amine oxidase, which forms FA from methylamine, have been implicated in disorders such as cerebrovascular disorders, alcohol abuse, diabetes and Alzheimer's disease. Phenelzine (PLZ), a monoamine oxidase inhibitor, is an antidepressant that has recently received attention for its neuroprotective/neurorescue properties. We investigated FA-induced toxicity and the effects of PLZ using rat primary cortical neurons and astrocytes and found that FA induced toxicity in neurons and astrocytes by multiple means. In astrocytes, FA decreased glutamate transporter expression, inhibiting glutamate uptake. PLZ reversed the decrease of glutamate uptake and the alteration of the second messengers, AKT and p38, induced by FA. PLZ alone affected the GLT-1 glutamate transporter in opposite directions in astrocytes and neurons. Thus, PLZ has multiple actions in neurons and astrocytes that may contribute to its neuroprotection.

Doxycycline inhibits proinflammatory cytokines but not acute cerebral cytogenesis after hypoxia-ischemia in neonatal rats.

BACKGROUND: Neonatal hypoxia-ischemia (HI) is a major cause of perinatal brain injury and is associated with a spectrum of neuropsychiatric disorders. Although very few treatment options are currently available, doxycycline (DOXY) has been reported to be neuroprotective in neontatal HI. Our objective was to investigate the effects of DOXY on neonatal brain development in normal and HI rat pups. We hypothesized that DOXY would inhibit microglial activation but that developmentally important processes, including cytogenesis and trophic responses, would not be impaired. METHODS: To investigate the putative neurodevelopmental consequences of DOXY administration in a clinically relevant animal model of HI, we performed a time-course analysis such that postnatal rat pups received DOXY (10mg/kg) or vehicle immediately before HI (n >or= 6). We then assessed cytogenesis, proinflammatory cytokines, brain-derived neurotrophic factor (BDNF) and matrix metalloproteinases regionally and longitudinally. RESULTS: We found that DOXY significantly inhibits neuroinflammation in the frontal cortex, striatum and hippocampus; decreases interleukin-1Beta (IL-1Beta) and tumour necrosis factor-alpha (TNF-alpha); and augments BDNF following HI. In addition, DOXY-treated pups have significantly fewer 2-bromo-5-deoxyuridine (BrdU)-positive cells in the subventricular zone 6 hours post-HI. However, DOXY does not persistently affect cytogenesis in the subventricular zone or dentate gyrus up to 7 days post-HI. The BrdU-positive cells not expressing markers for mature neurons colabel with nestin, an intermediate filament protein typical of neuronal precursors. LIMITATIONS: Our study investigates "acute" neurodevelopment over the first 7 days of life after HI injury. Further long-term investigations into adulthood are underway. CONCLUSION: Taken together, our results suggest the putative clinical potential of DOXY in the management of neonatal cerebral HI injury.

Brain-derived neurotrophic factor drives the changes in excitatory synaptic transmission in the rat superficial dorsal horn that follow sciatic nerve injury.

Peripheral nerve injury can promote neuropathic pain. The basis of the 'central sensitization' that underlies this often intractable condition was investigated using 14-20-day chronic constriction injury (CCI) of the sciatic nerve of 20-day-old rats followed by electrophysiological analysis of acutely isolated spinal cord slices. In addition, defined-medium organotypic spinal cord slice cultures were exposed for 5-6 days to brain-derived neurotrophic factor (BDNF, 200 ng ml(-1)) or to medium conditioned with activated microglia (aMCM). Since microglial activation is an early consequence of CCI, the latter manipulation allowed us to model the effect of peripheral nerve injury on the dorsal horn in vitro. Using whole-cell recording from superficial dorsal horn neurons, we found that both BDNF and CCI increased excitatory synaptic drive to putative excitatory 'radial delay' neurons and decreased synaptic excitation of inhibitory 'tonic islet/central' neurons. BDNF also attenuated synaptic excitation of putative GABAergic neurons identified by glutamic acid decarboxylase (GAD) immunoreactivity. Intrinsic neuronal properties (rheobase, input resistance and action potential discharge rates) were unaffected. Exposure of organotypic cultures to either BDNF or aMCM increased overall excitability of the dorsal horn, as seen by increased cytoplasmic Ca(2+) responses to 35 mm K(+) as monitored by confocal Fluo-4AM imaging. The effect of aMCM was attenuated by the recombinant BDNF binding protein TrkBd5 and the effect of BDNF persisted when GABAergic inhibition was blocked with SR95531. These findings suggest that CCI enhances excitatory synaptic drive to excitatory neurons but decreases that to inhibitory neurons. Both effects are mediated by nerve injury-induced release of BDNF from microglia.

Effects of sciatic nerve axotomy on excitatory synaptic transmission in rat substantia gelatinosa.

Injury or section of a peripheral nerve can promote chronic neuropathic pain. This is initiated by the appearance and persistence of ectopic spontaneous activity in primary afferent neurons that promotes a secondary, enduring increase in excitability of sensory circuits in the spinal dorsal horn ("central sensitization"). We have previously shown that 10-20 days of chronic constriction injury (CCI) of rat sciatic nerve produce a characteristic "electrophysiological signature" or pattern of changes in synaptic excitation of five different electrophysiologically defined neuronal phenotypes in the substantia gelatinosa of the dorsal horn. Although axotomy and CCI send different signals to the dorsal horn, we now find, using whole cell recording, that the "electrophysiological signature" produced 12-22 days after sciatic axotomy is quite similar to that seen with CCI. Axotomy thus has little effect on resting membrane potential, rheobase, current-voltage characteristics, or excitability of most neuron types; however, it does decrease excitatory synaptic drive to tonic firing neurons, while increasing that to delay firing neurons. Since many tonic neurons are GABAergic, whereas delay neurons do not contain gamma-aminobutyric acid, axotomy may reduce synaptic excitation of inhibitory neurons while increasing that of excitatory neurons. Further analysis of spontaneous and miniature (tetrodotoxin-resistant) excitatory postsynaptic currents is consistent with the possibility that decreased excitation of tonic neurons reflects loss of presynaptic contacts. By contrast, increased excitation of "delay" neurons may reflect increased frequency of discharge of presynaptic action potentials. This would explain how synaptic excitation of tonic cells decreases despite the fact that axotomy increases spontaneous activity in primary afferent neurons.

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.

Long-term actions of interleukin-1beta on delay and tonic firing neurons in rat superficial dorsal horn and their relevance to central sensitization.

BACKGROUND: Cytokines such as interleukin 1beta (IL-1beta) have been implicated in the development of central sensitization that is characteristic of neuropathic pain. To examine its long-term effect on nociceptive processing, defined medium organotypic cultures of rat spinal cord were exposed to 100 pM IL-1beta for 6-8 d. Interleukin effects in the dorsal horn were examined by whole-cell patch-clamp recording and Ca(2+) imaging techniques. RESULTS: Examination of the cultures with confocal Fluo-4 AM imaging showed that IL-1beta increased the change in intracellular Ca(2+) produced by exposure to 35-50 mM K+. This is consistent with a modest increase in overall dorsal horn excitability. Despite this, IL-1beta did not have a direct effect on rheobase or resting membrane potential nor did it selectively destroy any specific neuronal population. All effects were instead confined to changes in synaptic transmission. A variety of pre- and postsynaptic actions of IL-1beta were seen in five different electrophysiologically-defined neuronal phenotypes. In putative excitatory 'delay' neurons, cytokine treatment increased the amplitude of spontaneous EPSC's (sEPSC) and decreased the frequency of spontaneous IPSC's (sIPSC). These effects would be expected to increase dorsal horn excitability and to facilitate the transfer of nociceptive information. However, other actions of IL-1beta included disinhibition of putative inhibitory 'tonic' neurons and an increase in the amplitude of sIPSC's in 'delay' neurons. CONCLUSION: Since spinal microglial activation peaks between 3 and 7 days after the initiation of chronic peripheral nerve injury and these cells release IL-1beta at this time, our findings define some of the neurophysiological mechanisms whereby nerve-injury induced release of IL-1beta may contribute to the central sensitization associated with chronic neuropathic pain.

Postresuscitation N-acetylcysteine treatment reduces cerebral hydrogen peroxide in the hypoxic piglet brain.

OBJECTIVE: Reactive oxygen species have been implicated in the pathogenesis of hypoxia-reoxygenation injury. However, little information is known regarding the temporal profile of cerebral hydrogen peroxide (HPO) production and its response to N-acetylcysteine (an antioxidant) administration during neonatal hypoxia-reoxygenation. Using an acute swine model of neonatal hypoxia-reoxygenation, we examined the short-term neuroprotective effects of N-acetylcysteine on cerebral HPO production and oxidative stress in the brain. DESIGN: Controlled, block-randomized animal study. SETTING: University animal research laboratory. SUBJECTS: Newborn piglets (1-3 days, 1.7-2.1 kg). INTERVENTIONS: At 5 min after reoxygenation, piglets were given either saline or N-acetylcysteine (20 or 100 mg/kg/h) in a blinded, randomized fashion. MEASUREMENTS AND RESULTS: Newborn piglets were block-randomized into a sham-operated group (without hypoxia-reoxygenation, n = 5) and three hypoxic-reoxygenated groups (2 h of normocapnic alveolar hypoxia followed by 2h of reoxygenation, n = 7/group). Heart rate, mean arterial pressure, cortical HPO concentration, amino acid levels in cerebral microdialysate, and cerebral tissue glutathione and lipid hydroperoxide levels were examined. Hypoxic piglets were hypotensive and acidotic, and they recovered similarly in all hypoxic-reoxygenated groups. In hypoxic-reoxygenated control piglets, the cortical HPO concentration gradually increased during reoxygenation. Both doses of N-acetylcysteine abolished the increased HPO concentration and oxidized glutathione levels and tended to reduce the glutathione ratio and lipid hydroperoxide levels in the cerebral cortex (p = 0.08 and p = 0.1 vs. controls, respectively). N-acetylcysteine at 100mg/kg/h also increased the cerebral extracellular taurine levels. CONCLUSION: In newborn piglets with hypoxia-reoxygenation, postresuscitation administration of N-acetylcysteine reduces cerebral HPO production and oxidative stress, probably through a taurine-related mechanism.

Persistent neurochemical changes in neonatal piglets after hypoxia-ischemia and resuscitation with 100%, 21% or 18% oxygen.

BACKGROUND: Neonatal hypoxia-ischemia (HI) is a common complication of pregnancy and delivery. Conventional clinical practice is to resuscitate neonates with 100% O2, and evidence is building to suggest resuscitation with lower O2 concentrations is safer. Significant neurochemical changes are associated with HI injury and persistent changes in amino acids are related to cell death, therefore we used a swine survival model of neonatal HI-reoxygenation (HI/R) to investigate the effects of resuscitation with 100%, 21% or 18% O2 on amino acid neurotransmitters. METHODS: In a blinded randomized fashion, following permanent ligation of the left common carotid artery, newborn pigs (1-4 d, 1.7-2.5 kg) received alveolar normocapnic hypoxia (FiO2=0.15, 2h) and were reoxygenated with 18%, 21% or 100% O2. After a 4-day survival period, brain regions were processed for amino acid levels using high-performance liquid chromatography (HPLC). RESULTS: Results showed that resuscitation with different O2 concentrations caused hemispheric and regional changes in all amino acids investigated including glutamate, alanine, gamma-amino butyric acid, glycine and aspartate, 4 days post-HI. Resuscitation with 100% O2 significantly increased glutamate and glycine in the dorsal cortex contralateral to the ligated common carotid artery, compared to piglets resuscitated with 21% O2. Additionally, piglets resuscitated with 21% O2 had significantly lower alanine levels than those resuscitated with 18% O2. CONCLUSION: Significant resuscitation-dependent changes in amino acid neurotransmitters are still evident 4 days post-HI in the newborn piglet. These data suggest that persistent changes in neurochemistry occur 4 days after HI/R and further studies are warranted to elucidate the consequences of this on neonatal brain development.

Neurochemical effects of the monoamine oxidase inhibitor phenelzine on brain GABA and alanine: A comparison with vigabatrin.

PURPOSE: To compare phenelzine (PLZ), an antidepressant drug with anxiolytic properties which inhibits monoamine oxidase (MAO) but also elevates rat brain levels of the amino acids ?-aminobutyric acid (GABA) and alanine (ALA), with vigabatrin (VIG), an anticonvulsant which elevates brain GABA by inhibition of GABA transaminase (GABA-T), with regard to their actions on brain levels of GABA and ALA and on activities of MAO, GABA-T and ALA transaminase (ALA-T). METHODS: Male rats were administered PLZ (10 mg/kg) or VIG (1,000 mg/kg) i.p., and the rats were euthanized 4 hours later and the brains removed for analysis of levels of GABA and ALA (by electron capture gas chromatography after derivatization) and activities of MAO, GABA-T and ALA-T (radiochemical assays). RESULTS: Both PLZ and VIG inhibited GABA-T and elevated GABA levels. Only PLZ inhibited MAO and ALA-T and elevated ALA levels. The effects of PLZ on both amino acids and their transaminases were blocked by pre-treatment with the MAO inhibitor tranylcypromine. This pretreament had no effect on the inhibition of GABA-T or the elevation of brain GABA levels produced by VIG. CONCLUSIONS: At the doses studied, PLZ was as effective as VIG at elevating brain GABA levels, but, unlike VIG, also inhibited MAO and ALA-T (and increased brain ALA levels). Pretreatment of rats with the MAO inhibitor tranylcypromine prevented the increase in brain GABA and ALA levels with PLZ, but did not block the effect of VIG on GABA. These observations with tranylcypromine and PLZ support the hypothesis that an active metabolite of PLZ produced by the actions of MAO on this drug plays a major role in its GABA- and ALA-elevating actions.

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.