Neuroimmune characterization of optineurin insufficiency mouse model during ageing.

Optineurin is a multifunctional polyubiquitin-binding protein implicated in inflammatory signalling. Optineurin mutations are associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), neurodegenerative diseases characterised by neuronal loss, neuroinflammation, and peripheral immune disbalance. However, the pathogenic role of optineurin mutations is unclear. We previously observed no phenotype in the unmanipulated young optineurin insufficiency mice (Optn470T), designed to mimic ALS/FTD-linked truncations deficient in polyubiquitin binding. The purpose of this study was to investigate whether ageing would trigger neurodegeneration. We performed a neurological, neuropathological, and immunological characterization of ageing wild-type (WT) and Optn470T mice. No motor or cognitive differences were detected between the genotypes. Neuropathological analyses demonstrated signs of ageing including lipofuscin accumulation and microglial activation in WT mice. However, this was not worsened in Optn470T mice, and they did not exhibit TAR DNA-binding protein 43 (TDP-43) aggregation or neuronal loss. Spleen immunophenotyping uncovered T cell immunosenescence at two years but without notable differences between the WT and Optn470T mice. Conventional dendritic cells (cDC) and macrophages exhibited increased expression of activation markers in two-year-old Optn470T males but not females, although the numbers of innate immune cells were similar between genotypes. Altogether, a combination of optineurin insufficiency and ageing did not induce ALS/FTD-like immune imbalance and neuropathology in mice.

Chronic Pain-Associated Cardiovascular Disease: The Role of Sympathetic Nerve Activity.

Chronic pain affects many people world-wide, and this number is continuously increasing. There is a clear link between chronic pain and the development of cardiovascular disease through activation of the sympathetic nervous system. The purpose of this review is to provide evidence from the literature that highlights the direct relationship between sympathetic nervous system dysfunction and chronic pain. We hypothesize that maladaptive changes within a common neural network regulating the sympathetic nervous system and pain perception contribute to sympathetic overactivation and cardiovascular disease in the setting of chronic pain. We review clinical evidence and highlight the basic neurocircuitry linking the sympathetic and nociceptive networks and the overlap between the neural networks controlling the two.

Excessive dietary linoleic acid promotes plasma accumulation of pronociceptive fatty acyl lipid mediators.

Various fatty acyl lipid mediators are derived from dietary polyunsaturated fatty acids (PUFAs) and modulate nociception. The modern diet is rich in linoleic acid, which is associated with nociceptive hypersensitivities and may present a risk factor for developing pain conditions. Although recommendations about fatty acid intake exist for some diseases (e.g. cardiovascular disease), the role of dietary fatty acids in promoting pain disorders is not completely understood. To determine how dietary linoleic acid content influences the accumulation of pro- and anti-nociceptive fatty acyl lipid mediators, we created novel rodent diets using custom triglyceride blends rich in either linoleic acid or oleic acid. We quantified the fatty acyl lipidome in plasma of male and female rats fed these custom diets from the time of weaning through nine weeks of age. Dietary fatty acid composition determined circulating plasma fatty acyl lipidome content. Exposure to a diet rich in linoleic acid was associated with accumulation of linoleic and arachidonic acid-derived pro-nociceptive lipid mediators and reduction of anti-nociceptive lipid mediators derived from the omega-3 PUFAs. Our findings provide mechanistic insights into exaggerated nociceptive hypersensitivity associated with excessive dietary linoleic acid intake and highlight potential biomarkers for pain risk stratification.

Spinal Reflex Control of Arterial Blood Pressure: The Role of TRP Channels and Their Endogenous Eicosanoid Modulators.

The spinal cord is an important integrative center for blood pressure control. Spinal sensory fibers send projections to sympathetic preganglionic neurons of the thoracic spinal cord and drive sympathetically-mediated increases in blood pressure. While these reflexes responses occur in able-bodied individuals, they are exaggerated following interruption of descending control - such as occurs following spinal cord injury. Similar reflex control of blood pressure may exist in disease states, other than spinal cord injury, where there is altered input to sympathetic preganglionic neurons. This review primarily focuses on mechanisms wherein visceral afferent information traveling via spinal nerves influences sympathetic nerve activity and blood pressure. There is an abundance of evidence for the widespread presence of this spinal reflex arch originating from virtually every visceral organ and thus having a substantial role in blood pressure control. Additionally, this review highlights specific endogenous eicosanoid species, which modulate the activity of afferent fibers involved in this reflex, through their interactions with transient receptor potential (TRP) cation channels.

Purinergic receptor antagonism: A viable strategy for the management of autonomic dysreflexia?

The purinergic receptor ligand, ATP, may participate in reflex induced vasoconstriction through sympathetic efferent and sensory afferent mechanisms. However, the role of the purinergic system in contributing to autonomic dysreflexia following spinal cord injury is unclear. The present study investigates the involvement of P2X receptors in contributing to pressor responses during autonomic dysreflexia. Twenty rats were subjected to spinal cord injury and 24 h later hemodynamic responses to colorectal distension were recorded. Animals were randomized to receive intravenous administration of the P2X receptor antagonist, NF023, or vehicle control. The data indicate that NF023 attenuates pressor responses to colorectal distension.

Sleep disordered breathing induced by cervical spinal cord injury and effect of adenosine A1 receptors modulation in rats.

Sleep-disordered breathing (SDB) is very common after spinal cord injury (SCI). The present study was designed to evaluate the therapeutic efficacy of adenosine A1 receptor blockade (8-cyclopentyl-1,3-dipropylxanthine, DPCPX) on SDB in a rodent model of SCI. We hypothesized that SCI induced via left hemisection of the second cervical segment (C2Hx) results in SDB. We further hypothesized that blockade of adenosine A1 receptors following C2Hx would reduce the severity of SDB. In the first experiment, adult male rats underwent left C2Hx or sham (laminectomy) surgery. Unrestrained whole body plethysmography (WBP) and implanted wireless electroencephalogram (EEG) were used for assessment of breathing during spontaneous sleep and for the scoring of respiratory events at the acute (~1 wk), and chronic (~6 wk) time points following C2Hx. During the second experiment, the effect of oral administration of adenosine A1 receptor antagonist (DPCPX, 3 times a day for 4 days) on SCI induced SDB was assessed. C2Hx animals exhibited a higher apnea-hypopnea index (AHI) compared with the sham group, respectively (35.5 ± 12.6 vs. 19.1 ± 2.1 events/h, P < 0.001). AHI was elevated 6 wk following C2Hx (week 6, 32.0 ± 5.0 vs. week 1, 42.6 ± 11.8 events/h, respectively, P = 0.12). In contrast to placebo, oral administration of DPCPX significantly decreased AHI 4 days after the treatment (159.8 ± 26.7 vs. 69.5 ± 8.9%, P < 0.05). Cervical SCI is associated with the development of SDB in spontaneously breathing rats. Adenosine A1 blockade can serve as a therapeutic target for SDB induced by SCI.NEW & NOTEWORTHY The two key novel findings of our study included that 1) induced cervical spinal cord injury results in sleep-disordered breathing in adult rats, and 2) oral therapy with an adenosine A1 receptor blockade using DPCPX is sufficient to significantly reduce apnea-hypopnea index following induced cervical spinal cord injury.

Development of a decerebrate model for investigating mechanisms mediating viscero-sympathetic reflexes in the spinalized rat.

Autonomic dysreflexia (AD) often occurs in individuals living with spinal cord injury (SCI) and is characterized by uncontrolled hypertension in response to otherwise innocuous stimuli originating below the level of the spinal lesion. Visceral stimulation is a predominant cause of AD in humans and effectively replicates the phenotype in rodent models of SCI. Direct assessment of sympathetic responses to viscerosensory stimulation in spinalized animals is challenging and requires invasive surgical procedures necessitating the use of anesthesia. However, administration of anesthesia markedly affects viscerosensory reactivity, and the effects are exacerbated following spinal cord injury (SCI). Therefore, the major goal of the present study was to develop a decerebrate rodent preparation to facilitate quantification of sympathetic responses to visceral stimulation in the spinalized rat. Such a preparation enables the confounding effect of anesthesia to be eliminated. Sprague-Dawley rats were subjected to SCI at the fourth thoracic segment. Four weeks later, renal sympathetic nerve activity (RSNA) responses to visceral stimuli were quantified in urethane/chloralose-anesthetized and decerebrate preparations. Visceral stimulation was elicited via colorectal distension (CRD) for 1 min. In the decerebrate preparation, CRD produced dose-dependent increases in mean arterial pressure (MAP) and RSNA and dose-dependent decreases in heart rate (HR). These responses were significantly greater in magnitude among decerebrate animals when compared with urethane/chloralose-anesthetized controls and were markedly attenuated by the administration of urethane/chloralose anesthesia after decerebration. We conclude that the decerebrate preparation enables high-fidelity quantification of neuronal reactivity to visceral stimulation in spinalized rats. NEW & NOTEWORTHY In animal models commonly used to study spinal cord injury, quantification of sympathetic responses is particularly challenging due to the increased susceptibility of spinal reflex circuits to the anesthetic agents generally required for experimentation. This constitutes a major limitation to understanding the mechanisms mediating regionally specific neuronal responses to visceral activation in chronically spinalized animals. In the present study, we describe a spinalized, decerebrate rodent preparation that facilitates quantification of sympathetic reactivity in response to visceral stimuli following spinal cord injury. This preparation enables reliable and reproducible quantification of viscero-sympathetic reflex responses resembling those elicited in conscious animals and may provide added utility for preclinical evaluation of neuropharmacological agents for the management of autonomic dysreflexia.

Colocalization of A2a but not A1 adenosine receptors with GABA-ergic neurons in cardiopulmonary chemoreflex network in the caudal nucleus of the solitary tract.

Adenosine operating in the nucleus of the solitary tract (NTS) may inhibit or facilitate neurotransmitter release from nerve terminals and directly inhibit or facilitate central neurons via A1 and A2a pre- and postsynaptic receptors, respectively. However, adenosine A2a receptors, may also activate GABA-ergic neurons/terminals which in turn inhibit glutamatergic transmission in the NTS network. Our previous studies showed that adenosine operating via both A1 (inhibitor) and A2a (activator) receptors powerfully inhibits the cardiopulmonary chemoreflex (CCR) at the level of the caudal NTS. A1 receptors most likely inhibit glutamate release in the CCR network, whereas A2a receptors facilitate NTS GABA-ergic mechanisms which in turn inhibit CCR glutamatergic transmission. Therefore, we hypothesized that A2a receptors are located on NTS GABA-ergic neurons/terminals whereas A1 receptors may be located on NTS glutamatergic neurons/terminals. We investigated this hypothesis using double immunofluorescent staining for A2a or A1 adenosine receptors and GABA synthesizing enzyme, GAD67, in 30 µm thick, floating, medullary rat sections. We found that A2a adenosine receptors are localized within the GABA-ergic cells in the caudal NTS, whereas A1 adenosine receptors are absent from these neurons. Instead, A1 receptors were located on non-GABA-ergic (likely glutamatergic) neurons/terminals in the caudal NTS. These data support our functional findings and the hypothesis that adenosine A2a, but not A1 receptors are located on GABA-ergic neurons.

Nanoconjugate-bound adenosine A1 receptor antagonist enhances recovery of breathing following acute cervical spinal cord injury.

Respiratory complications in patients with spinal cord injury (SCI) are common and can have a negative impact on the quality of patients' lives. Previously, we found that intradiaphragmatic administration of the nanoconjugate-bound A1 adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) induced recovery of diaphragm function following SCI in rats. When administered immediately following the injury, recovery was observed as early as 3days following SCI and it persisted until the end of the study, 28days after the drug delivery. The recovery was observed using diaphragmatic electromyography (EMG) as well as phrenic nerve recordings; both of which were conducted under anesthetized conditions. Confounding effects of anesthetic may make data interpretation complex in terms of the impact on overall ventilatory function and clinical relevance. The objective of the present study was to test the hypothesis that intradiaphragmatic administration of nanoconjugate-bound DPCPX, enhances recovery of ventilation following SCI in the unanesthetized rat. To that end, Sprague-Dawley rats underwent C2 spinal cord hemisection (C2Hx) on day 0 and received either: (i) 0.15µg/kg of nanoconjugate-bound DPCPX or (ii) vehicle control (50µl distilled water). To assess ventilation, unrestrained whole body plethysmography (WBP) was performed on day 0 (immediately before the surgery) and 3, 7, 14, 21 and 28days following the SCI. Frequency, tidal volume, and minute ventilation data were analyzed in two minute bins while the animal was calm and awake. We found that a single administration of the nanoconjugate-bound A1 adenosine receptor antagonist facilitated recovery of tidal volume and minute ventilation following SCI. Furthermore, the treatment attenuated SCI-associated increases in respiratory frequency. Taken together, this study suggests that the previously observed DPCPX nanoconjugate-induced recovery in diaphragmatic and phrenic motor outputs may translate to a clinically meaningful improvement in ventilatory function in patients with SCI.

Transporter protein and drug-conjugated gold nanoparticles capable of bypassing the blood-brain barrier.

Drug delivery to the central nervous system (CNS) is challenging due to the inability of many drugs to cross the blood-brain barrier (BBB). Here, we show that wheat germ agglutinin horse radish peroxidase (WGA-HRP) chemically conjugated to gold nanoparticles (AuNPs) can be transported to the spinal cord and brainstem following intramuscular injection into the diaphragm of rats. We synthesized and determined the size and chemical composition of a three-part nanoconjugate consisting of WGA-HRP, AuNPs, and drugs for the treatment of diaphragm paralysis associated with high cervical spinal cord injury (SCI). Upon injection into the diaphragm muscle of rats, we show that the nanoconjugate is capable of delivering the drug at a much lower dose than the unconjugated drug injected systemically to effectively induce respiratory recovery in rats following SCI. This study not only demonstrates a promising strategy to deliver drugs to the CNS bypassing the BBB but also contributes a potential nanotherapy for the treatment of respiratory muscle paralysis resulted from cervical SCI.

Transporter Protein-Coupled DPCPX Nanoconjugates Induce Diaphragmatic Recovery after SCI by Blocking Adenosine A1 Receptors.

Respiratory complications in patients with spinal cord injury (SCI) are common and have a negative impact on the quality of patients' lives. Systemic administration of drugs that improve respiratory function often cause deleterious side effects. The present study examines the applicability of a novel nanotechnology-based drug delivery system, which induces recovery of diaphragm function after SCI in the adult rat model. We developed a protein-coupled nanoconjugate to selectively deliver by transsynaptic transport small therapeutic amounts of an A1 adenosine receptor antagonist to the respiratory centers. A single administration of the nanoconjugate restored 75% of the respiratory drive at 0.1% of the systemic therapeutic drug dose. The reduction of the systemic dose may obviate the side effects. The recovery lasted for 4 weeks (the longest period studied). These findings have translational implications for patients with respiratory dysfunction after SCI. SIGNIFICANCE STATEMENT: The leading causes of death in humans following SCI are respiratory complications secondary to paralysis of respiratory muscles. Systemic administration of methylxantines improves respiratory function but also leads to the development of deleterious side effects due to actions of the drug on nonrespiratory sites. The importance of the present study lies in the novel drug delivery approach that uses nanotechnology to selectively deliver recovery-inducing drugs to the respiratory centers exclusively. This strategy allows for a reduction in the therapeutic drug dose, which may reduce harmful side effects and markedly improve the quality of life for SCI patients.

NTS adenosine A2a receptors inhibit the cardiopulmonary chemoreflex control of regional sympathetic outputs via a GABAergic mechanism.

Adenosine is a powerful central neuromodulator acting via opposing A1 (inhibitor) and A2a (activator) receptors. However, in the nucleus of the solitary tract (NTS), both adenosine receptor subtypes attenuate cardiopulmonary chemoreflex (CCR) sympathoinhibition of renal, adrenal, and lumbar sympathetic nerve activity and attenuate reflex decreases in arterial pressure and heart rate. Adenosine A1 receptors inhibit glutamatergic transmission in the CCR pathway, whereas adenosine A2a receptors most likely facilitate release of an unknown inhibitory neurotransmitter, which, in turn, inhibits the CCR. We hypothesized that adenosine A2a receptors inhibit the CCR via facilitation of GABA release in the NTS. In urethane-chloralose-anesthetized rats (n = 51), we compared regional sympathetic responses evoked by stimulation of the CCR with right atrial injections of the 5-HT3 receptor agonist phenylbiguanide (1-8 µg/kg) before and after selective stimulation of NTS adenosine A2a receptors [microinjections into the NTS of CGS-21680 (20 pmol/50 nl)] preceded by blockade of GABAA or GABAB receptors in the NTS [bicuculline (10 pmol/100 nl) or SCH-50911 (1 nmol/100 nl)]. Blockade of GABAA receptors virtually abolished adenosine A2a receptor-mediated inhibition of the CCR. GABAB receptors had much weaker but significant effects. These effects were similar for the different sympathetic outputs. We conclude that stimulation of NTS adenosine A2a receptors inhibits CCR-evoked hemodynamic and regional sympathetic reflex responses via a GABA-ergic mechanism.

Severe hemorrhage attenuates cardiopulmonary chemoreflex control of regional sympathetic outputs via NTS adenosine receptors.

Selective stimulation of inhibitory A1 and facilitatory A2a adenosine receptor subtypes located in the nucleus of the solitary tract (NTS) powerfully inhibits cardiopulmonary chemoreflex (CCR) control of regional sympathetic outputs via different mechanisms: direct inhibition of glutamate release and facilitation of an inhibitory neurotransmitter release, respectively. However, it remains unknown whether adenosine naturally released into the NTS has similar inhibitory effects on the CCR as the exogenous agonists do. Our previous study showed that adenosine is released into the NTS during severe hemorrhage and contributes to reciprocal changes of renal (decreases) and adrenal (increases) sympathetic nerve activity observed in this setting. Both A1 and A2a adenosine receptors are involved. Therefore, we tested the hypothesis that, during severe hemorrhage, CCR control of the two sympathetic outputs is attenuated by adenosine naturally released into the NTS. We compared renal and adrenal sympathoinhibitory responses evoked by right atrial injections of 5HT3 receptor agonist phenylbiguanide (2-8 µg/kg) under control conditions, during hemorrhage, and during hemorrhage preceded by blockade of NTS adenosine receptors with bilateral microinjections of 8-(p-sulfophenyl) theophylline (1 nmol/100 nl) in urethane/chloralose anesthetized rats. CCR-mediated inhibition of renal and adrenal sympathetic activity was significantly attenuated during severe hemorrhage despite reciprocal changes in the baseline activity levels, and this attenuation was removed by bilateral blockade of adenosine receptors in the caudal NTS. This confirmed that adenosine endogenously released into the NTS has a similar modulatory effect on integration of cardiovascular reflexes as stimulation of NTS adenosine receptors with exogenous agonists.

Nucleus tractus solitarii A(2a) adenosine receptors inhibit cardiopulmonary chemoreflex control of sympathetic outputs.

Previously we have shown that stimulation of inhibitory A1 adenosine receptors located in the nucleus tractus solitarii (NTS) attenuates cardiopulmonary chemoreflex (CCR) evoked inhibition of renal, adrenal and lumbar sympathetic nerve activity and reflex decreases in arterial pressure and heart rate. Activation of facilitatory A2a adenosine receptors, which dominate over A1 receptors in the NTS, contrastingly alters baseline activity of regional sympathetic outputs: it decreases renal, increases adrenal and does not change lumbar nerve activity. Considering that NTS A2a receptors may facilitate release of inhibitory transmitters we hypothesized that A2a receptors will act in concert with A1 receptors differentially inhibiting regional sympathetic CCR responses (adrenal>lumbar>renal). In urethane/chloralose anesthetized rats (n=38) we compared regional sympathetic responses evoked by stimulation of the CCR with right atrial injections of serotonin 5HT3 receptor agonist, phenylbiguanide, (1-8µg/kg) before and after selective stimulation, blockade or combined blockade and stimulation of NTS A2a adenosine receptors (microinjections into the NTS of CGS-21680 0.2-20pmol/50nl, ZM-241385 40pmol/100nl or ZM-241385+CGS-21680, respectively). We found that stimulation of A2a adenosine receptors uniformly inhibited the regional sympathetic and hemodynamic reflex responses and this effect was abolished by the selective blockade of NTS A2a receptors. This indicates that A2a receptor triggered inhibition of CCR responses and the contrasting shifts in baseline sympathetic activity are mediated via different mechanisms. These data implicate that stimulation of NTS A2a receptors triggers unknown inhibitory mechanism(s) which in turn inhibit transmission in the CCR pathway when adenosine is released into the NTS during severe hypotension.

Activation of NTS A(1) adenosine receptors inhibits regional sympathetic responses evoked by activation of cardiopulmonary chemoreflex.

Previously we have shown that adenosine operating via the A(1) receptor subtype may inhibit glutamatergic transmission in the baroreflex arc within the nucleus of the solitary tract (NTS) and differentially increase renal (RSNA), preganglionic adrenal (pre-ASNA), and lumbar (LSNA) sympathetic nerve activity (ASNA>RSNA≥LSNA). Since the cardiopulmonary chemoreflex and the arterial baroreflex are mediated via similar medullary pathways, and glutamate is a primary transmitter in both pathways, it is likely that adenosine operating via A(1) receptors in the NTS may differentially inhibit regional sympathetic responses evoked by activation of cardiopulmonary chemoreceptors. Therefore, in urethane-chloralose-anesthetized rats (n = 37) we compared regional sympathoinhibition evoked by the cardiopulmonary chemoreflex (activated with right atrial injections of serotonin 5HT(3) receptor agonist phenylbiguanide, PBG, 1-8 µg/kg) before and after selective stimulation of NTS A(1) adenosine receptors [microinjections of N(6)-cyclopentyl adenosine (CPA), 0.033-330 pmol/50 nl]. Activation of cardiopulmonary chemoreceptors evoked differential, dose-dependent sympathoinhibition (RSNA>ASNA>LSNA), and decreases in arterial pressure and heart rate. These differential sympathetic responses were uniformly attenuated in dose-dependent manner by microinjections of CPA into the NTS. Volume control (n = 11) and blockade of adenosine receptor subtypes in the NTS via 8-(p-sulfophenyl)theophylline (8-SPT, 1 nmol in 100 nl) (n = 9) did not affect the reflex responses. We conclude that activation of NTS A(1) adenosine receptors uniformly inhibits neural and cardiovascular cardiopulmonary chemoreflex responses. A(1) adenosine receptors have no tonic modulatory effect on this reflex under normal conditions. However, when adenosine is released into the NTS (i.e., during stress or severe hypotension/ischemia), it may serve as negative feedback regulator for depressor and sympathoinhibitory reflexes integrated in the NTS.