Systems analysis shows that thermodynamic physiological and pharmacological fundamentals drive COVID-19 and response to treatment.

Why a systems analysis view of this pandemic? The current pandemic has inflicted almost unimaginable grief, sorrow, loss, and terror at a global scale. One of the great ironies with the COVID-19 pandemic, particularly early on, is counter intuitive. The speed at which specialized basic and clinical sciences described the details of the damage to humans in COVID-19 disease has been impressive. Equally, the development of vaccines in an amazingly short time interval has been extraordinary. However, what has been less well understood has been the fundamental elements that underpin the progression of COVID-19 in an individual and in populations. We have used systems analysis approaches with human physiology and pharmacology to explore the fundamental underpinnings of COVID-19 disease. Pharmacology powerfully captures the thermodynamic characteristics of molecular binding with an exogenous entity such as a virus and its consequences on the living processes well described by human physiology. Thus, we have documented the passage of SARS-CoV-2 from infection of a single cell to species jump, to tropism, variant emergence and widespread population infection. During the course of this review, the recurrent observation was the efficiency and simplicity of one critical function of this virus. The lethality of SARS-CoV-2 is due primarily to its ability to possess and use a variable surface for binding to a specific human target with high affinity. This binding liberates Gibbs free energy (GFE) such that it satisfies the criteria for thermodynamic spontaneity. Its binding is the prelude to human host cellular entry and replication by the appropriation of host cell constituent molecules that have been produced with a prior energy investment by the host cell. It is also a binding that permits viral tropism to lead to high levels of distribution across populations with newly formed virions. This thermodynamic spontaneity is repeated endlessly as infection of a single host cell spreads to bystander cells, to tissues, to humans in close proximity and then to global populations. The principal antagonism of this process comes from SARS-CoV-2 itself, with its relentless changing of its viral surface configuration, associated with the inevitable emergence of variants better configured to resist immune sequestration and importantly with a greater affinity for the host target and higher infectivity. The great value of this physiological and pharmacological perspective is that it reveals the fundamental thermodynamic underpinnings of SARS-CoV-2 infection.

The interacting physiology of COVID-19 and the renin-angiotensin-aldosterone system: Key agents for treatment.

SARS-CoV-2 interacting with its receptor, angiotensin-converting enzyme 2 (ACE2), turns the host response to viral infection into a dysregulated uncontrolled inflammatory response. This is because ACE2 limits the production of the peptide angiotensin II (Ang II) and SARS-CoV-2, through the destruction of ACE2, allows the uncontrolled production of Ang II. Recovery from trauma requires activation of both a tissue response to injury and activation of a whole-body response to maintain tissue perfusion. Tissue and circulating renin-angiotensin systems (RASs) play an essential role in the host response to infection and injury because of the actions of Ang II, mediated via its AT1 receptor. Both tissue and circulating arms of the renin angiotensin aldosterone system's (RAAS) response to injury need to be regulated. The effects of Ang II and the steroid hormone, aldosterone, on fluid and electrolyte homeostasis and on the circulation are controlled by elaborate feedback networks that respond to alterations in the composition and volume of fluids within the circulatory system. The role of Ang II in the tissue response to injury is however, controlled mainly by its metabolism and conversion to Ang-(1-7) by the enzyme ACE2. Ang-(1-7) has effects that are contrary to Ang II-AT1 R mediated effects. Thus, destruction of ACE2 by SARS-CoV-2 results in loss of control of the pro-inflammatory actions of Ang II and tissue destruction. Therefore, it is the response of the host to SARS-CoV-2 that is responsible for the pathogenesis of COVID-19.

"LONG COVID"-A hypothesis for understanding the biological basis and pharmacological treatment strategy.

Infection of humans with SARS-CoV-2 virus causes a disease known colloquially as "COVID-19" with symptoms ranging from asymptomatic to severe pneumonia. Initial pathology is due to the virus binding to the ACE-2 protein on endothelial cells lining blood vessels and entering these cells in order to replicate. Viral replication causes oxidative stress due to elevated levels of reactive oxygen species. Many (~60%) of the infected people appear to have eliminated the virus from their body after 28 days and resume normal activity. However, a significant proportion (~40%) experience a variety of symptoms (loss of smell and/or taste, fatigue, cough, aching pain, "brain fog," insomnia, shortness of breath, and tachycardia) after 12 weeks and are diagnosed with a syndrome named "LONG COVID." Longitudinal clinical studies in a group of subjects who were infected with SARS-CoV-2 have been compared to a non-infected matched group of subjects. A cohort of infected subjects can be identified by a battery of cytokine markers to have persistent, low level grade of inflammation and often self-report two or more troubling symptoms. There is no drug that will relieve their symptoms effectively. It is hypothesized that drugs that activate the intracellular transcription factor, nuclear factor erythroid-derived 2-like 2 (NRF2) may increase the expression of enzymes to synthesize the intracellular antioxidant, glutathione that will quench free radicals causing oxidative stress. The hormone melatonin has been identified as an activator of NRF2 and a relatively safe chemical for most people to ingest chronically. Thus, it is an option for consideration of re-purposing studies in "LONG COVID" subjects experiencing insomnia, depression, fatigue, and "brain fog" but not tachycardia. Appropriately designed clinical trials are required to evaluate melatonin.

Discovery of N-Aryloxypropylbenzylamines as Voltage-Gated Sodium Channel NaV 1.2-Subtype-Selective Inhibitors.

We previously reported that a lipophilic N-(4'-hydroxy-3',5'-di-tert-butylbenzyl) derivative (1) of the voltage-gated sodium channel blocker mexiletine, was a more potent sodium channel blocker in vitro and in vivo. We demonstrate that replacing the chiral methylethylene linker between the amine and di-tert-butylphenol with an achiral 1,3-propylene linker (to give (2)) maintains potency in vitro. We synthesized 25 analogues bearing the 1,3-propylene linker and found that minor structural changes resulted in pronounced changes in state dependence of blocking human NaV 1.2 and 1.6 channels by high-throughput patch-clamp analysis. Compared to mexiletine, compounds 1 and 2 are highly selective NaV 1.2 inhibitors and >500 times less potent in inhibiting NaV 1.6 channels. On the other hand, a derivative (compound 4) bearing 2,6-dimethoxy groups in place of the 2,6-dimethyl groups found in mexiletine was found to be the most potent inhibitor, but is nonselective against both channels in the tonic, frequency-dependent and inactivated states. In a kindled mouse model of refractory epilepsy, compound 2 inhibited seizures induced by 6 Hz 44 mA electrical stimulation with an IC50 value of 49.9±1.6 mg kg-1 . As established sodium channel blockers do not suppress seizures in this mouse model, this indicates that 2 could be a promising candidate for treating pharmaco-resistant epilepsy.

Tacrine: In vivo veritas.

Tacrine was initially synthesised in 1945 as part of a project seeking antibacterial drugs to treat infected wounds in soldiers. However, it was inactive in vitro against common strains of bacteria. Serendipitously, it was injected in vivo into dogs anaesthetised with chloroform and morphine and noted to immediately counter the respiratory rate depression caused by morphine but not block analgesia. Subsequent studies showed that tacrine was an acetylcholinesterase inhibitor. When combined with morphine in ampoules it was possible to inject larger doses of morphine without causing respiratory depression and it was marketed for 10 years in Australia. Tacrine was also used alone for treating acute anticholinergic syndrome in the 1980s. Shortly after this, it was hypothesised by William Summers that it could be of benefit in treating the early stages of Alzheimer's dementia and an IND was granted by the US Food and Drug Administration and a use patent awarded to Summers. It was the first of four anticholinesterases to be approved for treating this condition although its variable pharmacokinetics was a disadvantage.

Chronic Brain Inflammation: The Neurochemical Basis for Drugs to Reduce Inflammation.

It is now recognised that the brain and the peripheral immune system have bidirectional communication in both health and neuronal diseases. Brain inflammation results after both acute injury and also with the appearance of mutated proteins or endogenous neurotoxic metabolites associated with slow neurodegenerative diseases such as Alzheimer's and Parkinson's diseases and some psychiatric disorders. Microglia play a key role in brain inflammation by the release of pro-inflammatory cytokines and with ageing, microglia exhibit 'priming' leading to increased basal release of the pro-inflammatory cytokines. Neurochemical targets to reduce or slow chronic brain inflammation include cyclooxygenase enzymes, Nrf2 transcription factor, angiotensin AT1 receptors and sigma-1 receptors. Development of more selective drugs to act at these targets is occurring but large scale clinical trials to validate the drugs will take significant time.

Computational Analysis of Amiloride Analogue Inhibitors of Coxsackievirus B3 RNA Polymerase.

Coxsackievirus B3 (CVB3) is a picornavirus that is responsible for a significant proportion of human myocarditis. However, no antiviral treatment is currently available to treat this disease or indeed any picornaviral infections. Previously it was shown that amiloride and its derivative 5-(N-ethyl-N-isopropyl)amiloride inhibit the in vitro enzymatic activity of CVB3 RNA polymerase (3Dpol). Here we measure and compare the inhibitory activity of ten amiloride analogues against CVB3 3Dpol. We show that replacement of the 3,5-diaminopyrazinyl moiety of amiloride causes loss of the inhibitory activity, whereas modifications at the 5-amino and guanidino groups increase or decrease potency. Importantly, a combination of substitutions at both the 5-amino and guanidino groups produced a compound that was more potent than its singly modified precursors. The compounds were computationally-docked into available crystal structures of CVB3 3Dpol in order to obtain a structural explanation for the activities of the analogues. To create a robust model which explained the biological activity, optimization of one of the CVB3 3Dpol crystal structures to take into account active site flexibility was necessary, together with the use of consensus docking from two different docking algorithms. This robust predictive 3D atomic model provides insights into the interactions required for inhibitor binding and provides a promising basis for the development of more potent inhibitors against this important therapeutic target.

An improved method to prepare an injectable microemulsion of the galanin-receptor 3 selective antagonist, SNAP 37889, using Kolliphor(®) HS 15.

Research into the galanin-3 (GAL3) receptor has many challenges, including the lack of commercially available selective ligands. While the identification of non-peptidergic GAL3 receptor-selective antagonists, 1-phenyl-3-[3-(trifluoromethyl)phenyl]iminoindol-2-one (SNAP 37889) and 1-[3-(2-pyrrolidin-1-ylethoxy)phenyl]-3-[3-(trifluoromethyl)phenyl]iminoindol-2-one (SNAP 398299) have implicated a role for GAL3 receptors in anxiety, depression and drug-seeking behaviour, a major limitation of their use is poor aqueous solubility. Previously we have used 5% dimethylsulfoxide (DMSO) with 1% hydroxypropylmethyl cellulose in saline to dissolve SNAP 37889 for intraperitoneal (i.p.) injections of rats; however this produced a micro-suspension that was not ideal. The injectable formulation of SNAP 37889 was improved as follows:•30% (w/v) Kolliphor(®) HS 15 (Solutol HS(®) 15) and sodium phosphate buffer (0.01 M, pH 7.4) were used as vehicles.•A smooth glass mortar and pestle was used to triturate the Kolliphor(®) HS 15 and SNAP 37889 into a paste before addition to the sodium phosphate buffer at room temperature (RT).•The resulting mixture was vortexed until the paste was fully dissolved and the microemulsion was allowed to sit for 20 min to allow air bubbles to coalesce.

Sigma-1 receptor ligand PRE-084 reduced infarct volume, neurological deficits, pro-inflammatory cytokines and enhanced anti-inflammatory cytokines after embolic stroke in rats.

Sigma receptor agonists have been found to provide potent neuroprotection in rats and mice. This neuroprotection is thought to be mediated through anti-excitotoxic mechanisms. Neuroprotective and immune modulatory effects of sigma ligands have not been investigated in embolic stroke. In the present study, rats were subjected to embolic stroke or sham stroke and were treated with the sigma-1 receptor agonist PRE-084 (5mg/kg i.p.) or saline vehicle 3 and 24h after stroke. Infarct volume and behavioural tests were conducted, and cytokine levels (ILs-1α and ß, IL-2, IL-4, IL-6, IL-10, GM-CSF and TNF-α) were determined in ischemic and non-ischemic cortices. Axonal damage was determined using the pNF-H ELISA assay. Treatment with PRE-084 afforded neuroprotection following embolic stroke as evidenced by significantly reduced infarct volume and improved behavioural outcome. Remarkably, treatment with PRE-084 reduced levels of pro-inflammatory cytokines and enhanced anti-inflammatory cytokines. Levels of pNF-H were lower in rats treated with PRE-084 suggesting reduced axonal damage but this finding did not reach statistical significance. The findings of the present study suggest that part of the neuroprotective effects of sigma-1 receptor agonists may be mediated through a dual effect on cytokine release following stroke.

Design and assessment of a potent sodium channel blocking derivative of mexiletine for minimizing experimental neuropathic pain in several rat models.

Physical or chemical damage to peripheral nerves can result in neuropathic pain which is not easily alleviated by conventional analgesic drugs. Substantial evidence has demonstrated that voltage-gated Na+ channels in the membrane of damaged nerves play a key role in the establishment and maintenance of pathological neuronal excitability not only of these peripheral nerves but also in the second- and third-order neurons in the pain pathway to the cerebral cortex. Na+ channel blocking drugs have been used in treating neuropathic pain with limited success mainly because of a preponderance of side-effects. We have developed an analogue of mexiletine which is approximately 80 times more potent than mexiletine in competing with the radioligand, 3H-batrachotoxinin for binding to Na+ channels in rat brain membranes and also it is much more lipophilic than mexiletine which should enhance its uptake into the brain to block the increased expression of Na+ channels on second- and third-order neurons of the pain pathway. This analogue, HFI-1, has been tested in three different rat models of neuropathic pain (formalin paw model, ligated spinal nerve model and contusive spinal cord injury model) and found to be more effective in reducing pain behaviours than mexiletine.

Meloxicam reduces lipopolysaccharide-induced degeneration of dopaminergic neurons in the rat substantia nigra pars compacta.

Inflammation is believed to play an important role in the etiology and pathogenesis of Parkinson's disease (PD). However, experimental and epidemiological evidences from various non-steroidal anti-inflammatory drugs, including cyclooxygenase-2 (COX-2) inhibitors, seem contradictive. Using the intranigral lipopolysaccharide (LPS) rat model, we show that meloxicam, a preferential COX-2 inhibitor, diminishes the activation of OX-42-immunoreactive (ir) microglia and reduces the loss of tyrosine hydroxylase (TH)-ir dopamine (DA) neurons in the substantia nigra pars compacta (SNpc) that is normally induced by exposure to LPS. Double-labelling immunohistochemistry identified that activated microglia rather than intact resting microglia are the main intracellular venues for COX-2 expression. These findings suggest that inhibition of COX-2 activity in activated microglial cells may be potentially neuroprotective for DA neurons in the SNpc.

Synthesis and evaluation of dithiolethiones as novel cyclooxygenase inhibitors.

3H-1,2-Dithiole-3-thiones substituted with a 3,5-di-tert-butyl-4-hydroxyphenyl (DTBHP) or a 3,5-di-tert-butyl-4-methoxyphenyl group at the C5 position were prepared and their ability to inhibit the cyclooxygenase isoenzymes, COX-1 and COX-2 was evaluated. Both compounds were potent inhibitors of COX-2 (relative to rofecoxib), and while the phenol was a weak inhibitor of COX-1, the methyl ether gave no measurable inhibition. Docking studies of the two compounds into the COX-1 and -2 active sites showed that the methyl ether could only fit in the COX-2 active site whereas the phenol could be docked into both COX-1 and -2. This study reports a new mode for inhibitor binding to COX-1 and -2 and a novel structural scaffold for the development of COX-2 selective inhibitors.

Validation of a novel biomarker for acute axonal injury in experimental autoimmune encephalomyelitis.

In multiple sclerosis, inflammatory axonal injury is a key pathological mechanism responsible for the development of progressive neurological dysfunction. The injured axon represents a therapeutic target in this disease; however, therapeutic trials of neuroprotective candidates will initially require preclinical testing in an animal model of inflammatory axonal injury and subsequently the development of a reliable paraclinical measure of axonal degeneration in humans. In the present study, we demonstrate the validity of serum phosphorylated neurofilament H (pNF-H) as a marker of axonal injury in murine experimental autoimmune encephalomyelitis (EAE). At the time of maximum disease severity (EAE day 22), the average serum pNF-H level reached 5.7 ng/ml, correlating significantly with the EAE paraplegia score (r = 0.75, P < 0.001). On average, 40% of axons in the spinal cord were lost in EAE, and serum pNF-H levels were highly correlated with axon loss (r = 0.8, P < 0.001). Axonal injury was a severe and acute event, insofar as serum pNF-H levels were not significantly elevated at early (EAE day 12) or late (EAE days 35 and 50) disease time points. Our results demonstrate that acute inflammatory axonal injury is a pathological feature of murine MOG(35-55) EAE, indicating that this model may mirror the acute pathological events in active multiple sclerosis lesions. Furthermore, we have validated the serum pNF-H assay as an unbiased measurement of axonal injury in EAE, facilitating rapid screening of potential neuroprotective therapies in this model.

Using behaviour to predict stroke severity in conscious rats: post-stroke treatment with 3', 4'-dihydroxyflavonol improves recovery.

Prognostic models are used to predict outcome in stroke patients and to stratify treatment groups in clinical trials. No one has previously attempted to use such models in stroke recovery studies in animals. We have now shown the predictive value of assigning stroke severity ratings, based on behaviours displayed in conscious rats during infusion of endothelin-1 to constrict the middle cerebral artery, on neurological and histological outcomes. The validity of prior stratification of treatment groups according to stroke ratings was tested by assessment of the protective potential of synthetic flavonol, 3',4'-dihydroxyflavonol (DiOHF). Neurological deficits and performance on the sticky label test were evaluated before and at 24, 48 and 72 h post-stroke. Histopathology was assessed at 72 h. Positive correlations between stroke ratings and neurological deficit scores were found at 24 (r=0.58, P<0.001), 48 (r=0.53, P<0.001) and 72 (r=0.56, P<0.001) h post-stroke, with more severe strokes associated with worse deficit scores. Similar correlations were observed with the sticky label test. Higher stroke ratings also correlated with greater infarct volumes (total infarct volume: r=0.74, P<0.0001). Treatment with DiOHF (10 mg/kg i.v. given 3, 24 and 48 h post-stroke) significantly reduced infarct volume and restored neurological function in rats with modest stroke ratings (P<0.01), but not in rats with high stroke ratings. These results suggest that stroke ratings, based on behavioural assessment as the stroke develops, reliably predict histopathological and functional outcomes and allow stratification of treatment groups. DiOHF given after stroke improves outcomes in moderate strokes, and therefore has cytoprotective potential.

Pharmacokinetics and brain uptake of AM-36, a novel neuroprotective agent, following intravenous administration to rats.

The plasma pharmacokinetics and brain uptake of the novel neuroprotective agent AM-36 (1-(2-(4-chlorophenyl)-2-hydroxy)ethyl-4-(3,5-bis-(1,1dimethylethyl)-4-hydroxyphenyl) methylpiperazine) were assessed over 72 h following i.v. administration to male Sprague-Dawley rats. At nominal i.v. doses of 0.2, 1 and 3mg kg(-1), AM-36 exhibited an extremely large volume of distribution (18.2-24.6 L kg(-1)) and a long terminal elimination half-life, ranging from 25.2 to 37.7 h. Over this dose range, AM-36 exhibited linear pharmacokinetics, with no apparent change in clearance, volume of distribution or dose-normalised area under the plasma concentration - time curve. AM-36 was very highly bound to plasma proteins (> 99.6%); however, this did not appear to affect the ability of AM-36 to permeate the blood-brain barrier. Following a single i.v. dose of AM-36 at 3mg kg(-1) to rats, brain concentrations were detected for up to 72 h, and the brain-to-plasma ratios were high at all time points (ranging from 8.2 at 5 min post-dose to 0.9 at 72 h post-dose). The very high brain uptake of AM-36 supports previous in-vivo efficacy studies demonstrating the neuroprotective effects of this compound when administered to rats with middle cerebral artery occlusion.

Inflammatory cell infiltration after endothelin-1-induced cerebral ischemia: histochemical and myeloperoxidase correlation with temporal changes in brain injury.

Accumulation of neutrophils in brain after transient focal stroke remains controversial with some studies showing neutrophils to be deleterious, whereas others suggest neutrophils do not contribute to ischemic injury. Myeloperoxidase (MPO) has been used extensively as a marker for quantifying neutrophil accumulation, but is an indirect method and does not detect neutrophils alone. To elucidate the interaction of macrophages in the neutrophil inflammatory response, we conducted double-label immunofluorescence in brain sections at 0, 1, 2, 3, 7, and 15 days after ischemia. Each of these results was obtained from the same animal to determine correlations between neutrophil infiltration and ischemic damage. It was found that MPO activity increased up to 3 days after cerebral ischemia. Dual-staining revealed that macrophages engulf neutrophils in the brain and that this engulfment of neutrophils increased with time, with 50% of neutrophils in the brain engulfed at 3 days and approximately 85% at 15 days (N=5, P<0.05). Interestingly, at 7 days the amount of dual-staining was decreased to 20% (N=5, P<0.05). Neutrophil infiltration was positively correlated with ischemic damage in both the cortex and striatum (r(2)=0.86 and 0.80, respectively, P<0.01). The results of this study indicate that the MPO from neutrophils phagocytized by macrophages may continue to contribute to the overall MPO activity, and that previous assessments that have utilized this marker to measure neutrophil accumulation may have mis-calculated the number of neutrophils within the ischemic territory and hence their contribution to the evolution of the infarct at later time points. Thus any biphasic infiltration of neutrophils may have been masked by the accumulation of macrophages.

Injury to axons and oligodendrocytes following endothelin-1-induced middle cerebral artery occlusion in conscious rats.

Injury to axons and oligodendrocytes has been poorly characterized in most animal models of stroke, and hence has been difficult to target therapeutically. It is therefore necessary to characterize axonal and oligodendroglial injury in these models, in order to rationally design putative protective compounds that minimize this injury. This study aims to characterize injury to axons and oligodendrocytes in the endothelin-1 (ET-1) model of middle cerebral artery occlusion (MCAO) in conscious rats. Transient forebrain ischemia was induced in conscious adult male Long Evans rats by the perivascular microinjection of ET-1. Quantitative histopathology was performed on forebrain sections at 6, 24, 48 and 72 h after ET-1 administration, using ballistic light analyses and immunohistochemistry for amyloid precursor protein (APP), SMI32, and Tau-1. Ballistic light analyses of cortical and striatal lesions revealed that the infarct volume was maximal in these regions by 6 h. APP and SMI32 immunohistochemistry demonstrated that axonal injury was maximal by 6 h in this model; however, some injured axons appeared to maintain good structural integrity up to 72 h after insult. Density measurements for Tau-1-immunopositive oligodendrocytes were significantly elevated within the corpus callosum from 48 h, but reductions in total oligodendrocyte numbers were not apparent up 72 h after ET-1 injection. These results indicate that axonal and oligodendroglial injury should be investigated as potential targets for delayed therapeutic intervention after MCAO.

Hypoxic preconditioning produces differential expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and its regulatory enzyme HIF prolyl hydroxylase 2 in neonatal rat brain.

Hypoxic preconditioning can protect the brain against a subsequent damaging ischaemic insult. Mild hypoxia alone seems not sufficient to cause neuronal injury, but can induce changes in gene expression and intracellular signalling pathways and the hypoxia-inducible transcription factor (HIF-1) is a key modulator of these genes. Recently, a family of HIF prolyl hydroxylase enzymes (PHDs) has been shown to regulate HIF-1 function by controlling its degradation. Since PHD-2 is thought to be the predominant isoform which regulates HIF-1, we have investigated whether preconditioning with hypoxia can affect levels of PHD-2 and HIF-1alpha proteins to elucidate roles for the HIF-1/PHD-2 system in the neuroprotection conferred by hypoxic preconditioning. Sprague-Dawley rats (postnatal Day 6 (p6)) were exposed to preconditioning with hypoxia (3 h, 8% oxygen) or normoxia (3 h, room air) at various times (0, 0.5, 2, 4, 16 and 24 h) after reoxygenation, brains were obtained for Western blot and immunohistochemical analyses of PHD-2 and HIF-1alpha proteins. Western blotting studies demonstrate a significant increase in the expression of PHD-2 ( approximately 1.8-2-fold increase, at 0.5, 16 and 24 h after reoxygenation; p < 0.01) and HIF-1alpha (approximately 1.7-fold increase immediately after hypoxia; p < 0.05) proteins following hypoxic preconditioning relative to normoxic control tissue. Similar results were observed in immunohistochemical studies examining PHD-2 and HIF-1alpha proteins. Our study demonstrated for the first time that in vivo exposure to systemic hypoxia elevates the expression of PHD-2 protein in brain and it is likely that enhancing HIF-1 function by inhibition of PHD activity is involved in the protective effect conferred by hypoxic preconditioning in neonatal rat brain.

Effects of lipopolysaccharide on glial phenotype and activity of glutamate transporters: Evidence for delayed up-regulation and redistribution of GLT-1.

Excitatory amino acid transporters (EAATs) are responsible for homeostasis of extracellular L-glutamate, and the glial transporters are functionally dominant. EAAT expression or function is altered in acute and chronic neurological conditions, but little is known about the regulation of EAATs in reactive astroglia found in such neuropathologies. These studies examined the effects of the bacterial endotoxin lipopolysaccharide (LPS) on glial EAATs in vitro. The effects of LPS (1 microg/ml, 24-72 h) on EAAT activity and expression were examined in primary cultures of mouse astrocytes. [(3)H]D-aspartate uptake increased to 129% of control by 72 h treatment with LPS. Saturation analysis revealed that apparent K(m) was unchanged whilst V(max) was significantly increased to 172% of control by 72 h LPS treatment. Biotinylation and Western blotting indicated that cell-surface expression of GLT-1 was significantly elevated (146% control) by LPS treatment whereas GLAST expression was unchanged. Confocal analyses revealed that LPS treatment resulted in cytoskeletal changes and stellation of astrocytes, with rearrangement of F-actin (as shown by phalloidin labelling). Immunocytochemistry revealed clustering of GLAST, and increased expression and redistribution of GLT-1 to the cell-surface following treatment with LPS. Similar experiments were conducted in microglia, where LPS (50 ng/ml) was found to up-regulate expression of GLT-1 at 24 and 72 h in concert with cytoskeletal changes accompanying activation. These findings suggest an association of cytoskeletal changes in glia with EAAT activity, with the predominant adaptation involving up-regulation and redistribution of GLT-1.

Localization of AT(2) receptors in the nucleus of the solitary tract of spontaneously hypertensive and Wistar Kyoto rats using [125I] CGP42112: upregulation of a non-angiotensin II binding site following unilateral nodose ganglionectomy.

We have examined the binding distribution of a selective AT(2) receptor ligand [125I] CGP42112 in the brain of adult Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). AT(2) receptor localization was also examined in the rat brainstem following unilateral nodose ganglionectomy. Specific [125I] CGP42112 binding was observed in discrete brain regions from both rat strains, including the nucleus of the solitary tract (NTS), and did not differ between WKY and SHR. [125I] CGP42112 binding in the NTS revealed an AT(2) receptor component that was displaceable by PD 123319 and Ang II (50-58%), as well as a non-angiotensin II receptor component (42-49%). Following unilateral nodose ganglionectomy, [125I] CGP42112 binding density on the denervated side of the NTS was increased approximately two-fold in both WKY and SHR. This increased [125I] CGP42112 binding density in the ipsilateral NTS was comprised of a greater non-angiotensin II component than that observed in the sham groups, since only approximately 30% was displaced by PD123319 and angiotensin II. Furthermore, [125I] CGP42112 also revealed high binding density on the denervated side in the dorsal motor nucleus and the nucleus ambiguus in both WKY and SHR. AT(2) receptor immunoreactivity was also visualised in the NTS of sham operated rats, but was not observed in the dorsal motor nucleus or the nucleus ambiguus, nor was it up-regulated following nodose ganglionectomy. These results demonstrate, for the first time, an AT(2) receptor binding site in the NTS, as well as a non-angiotensin II [125I] CGP42112 binding site. These studies also demonstrate that nodose ganglionectomy represents a useful model in which to study a non-angiotensin II [125I] CGP42112 binding site that is up-regulated following degeneration of afferent vagal nerves.