Abstract
Global cerebral ischemia following cardiac arrest and cardiopulmonary resuscitation (CA/CPR) causes injury to hippocampal CA1 pyramidal neurons and impairs cognition. Small conductance Ca
2+
...‐activated potassium channels type 2 (SK2), expressed in CA1 pyramidal neurons, have been implicated as potential protective targets. Here we showed that, in mice, hippocampal long‐term potentiation (LTP) was impaired as early as 3 h after recovery from CA/CPR and LTP remained impaired for at least 30 days. Treatment with the SK2 channel agonist 1‐Ethyl‐2‐benzimidazolinone (1‐EBIO) at 30 min after CA provided sustained protection from plasticity deficits, with LTP being maintained at control levels at 30 days after recovery from CA/CPR. Minimal changes in glutamate release probability were observed at delayed times after CA/CPR, implicating post‐synaptic mechanisms. Real‐time quantitative reverse transcriptase‐polymerase chain reaction indicated that CA/CPR did not cause a loss of
N
‐methyl‐D‐aspartate (NMDA) receptor
mRNA
at 7 or 30 days after CA/CPR. Similarly, no change in synaptic NMDA receptor protein levels was observed at 7 or 30 days after CA/CPR. Further, patch‐clamp experiments demonstrated no change in functional synaptic NMDA receptors at 7 or 30 days after CA/CPR. Electrophysiology recordings showed that synaptic SK channel activity was reduced for the duration of experiments performed (up to 30 days) and that, surprisingly, treatment with 1‐EBIO did not prevent the CA/CPR‐induced loss of synaptic SK channel function. We concluded that CA/CPR caused alterations in post‐synaptic signaling that were prevented by treatment with the SK2 agonist 1‐EBIO, indicating that activators of SK2 channels may be useful therapeutic agents to prevent ischemic injury and cognitive impairments.
Global cerebral ischemia following cardiac arrest and cardiopulmonary resuscitation (CA/CPR) causes injury to hippocampal CA1 pyramidal neurons and impairs cognition. SK2 channels, expressed in CA1 ...pyramidal neurons, have been implicated as potential protective targets. Here we show that in mice, hippocampal long-term potentiation (LTP) is impaired as early as 3 hrs after recovery from CA/CPR and that LTP remains impaired for at least 30 days. Treatment with the SK2 channel agonist, 1-EBIO 30 minutes after CA provided sustained protection from plasticity deficits, with LTP being maintained at control levels at 30 days after recovery from CA/CPR. Minimal changes in glutamate release probability were observed at delayed times after CA/CPR, implicating post-synaptic mechanisms. Real-time quantitative RT-PCR indicates that CA/CPR does not cause a loss of NMDA receptor mRNA 7 or 30 days after CA/CPR. Similarly, no change in synaptic NMDA receptor protein levels were observed 7 or 30 days after CA/CPR. Further, patch-clamp experiments demonstrate no change in functional synaptic NMDA receptors 7 or 30 days after CA/CPR. Electrophysiology recordings showed that synaptic SK channel activity is reduced for the duration of experiments performed (up to 30 days) and that surprisingly, treatment with 1-EBIO did not prevent CA/CPR-induced loss of synaptic SK channel function. We conclude that CA/CPR causes alterations in post-synaptic signaling that are prevented by treatment with the SK2 agonist 1-EBIO, indicating that activators of SK2 channels may be useful therapeutic agents to prevent ischemic injury and cognitive impairments.
Arrestins constitute a family of small cytoplasmic proteins that mediate deactivation of G-protein-coupled receptors (GPCRs) and are known to be essential for cascade inactivation and receptor ...desensitization. Alternative splicing produces an array of arrestin gene products that have widely different specificities for their cognate receptors in vitro, but the differential functions of these splice variants in vivo are essentially unknown. Bovine rod photoreceptors express two splice variants of visual arrestin (p44 and p48) that display different affinities for the GPCR rhodopsin. To determine the functions of these splice variants in intact cells, we expressed a transgene encoding either a truncated form of murine arrestin (mArr(1-369), or m44) or the long (p48) isoform in mouse rods lacking endogenous arrestin (Arr-/-). Morphological analysis showed that expression of either variant attenuated the light-induced degeneration that is thought to result from excessive cascade activity in Arr-/-rods. Suction electrode recordings from individual rods indicated that the expression of either m44 or p48 splice variants could restore normal kinetics to Arr-/- dim flash responses, indicating that both isoforms can bind to and quench phosphorylated rhodopsin rapidly. To our surprise, only the full-length variant was able to alter the kinetics of responses in rods lacking both arrestin and rhodopsin kinase, indicating that p48 can also quench the activity of nonphosphorylated rhodopsin.
Systemic sclerosis (SSc) is a multisystem autoimmune rheumatic disorder with high morbidity and the highest case specific mortality of the rheumatic diseases. There is no currently approved ...unequivocally effective treatment for SSc and therefore there is a huge unmet medical need for novel and effective therapies. Hyperimmune caprine serum (HCS) is a goat serum extract derivative produced from goats vaccinated with a detergent-inactivated HIV viral lysate. It contains caprine immunoglobulins and small molecular weight proteins as well as a CRH, α-2 macroglobulin (α-2M) and lipoprotein-related peptide-1 complex. In this thesis we explore the hypothesis that hyperimmune caprine serum improves skin and other measures of disease severity in established dcSSc by modulating immunological function that determines persistence of clinical disease. This hypothesis is explored through 1) a prospective clinical trial, 2) long-term clinical use and 3) detailed assessment of serum growth factors and cytokines, as well as established and exploratory markers of disease. The primary objective of the clinical trial was to explore safety and tolerability of HCS in established diffuse cutaneous systemic sclerosis (dcSSc). Secondary objectives included assessment of potential efficacy and biological activity and exploration of candidate biomarkers. There were no safety concerns and frequency of adverse events was not different between HCS and placebo group. MRSS improved in the HCS group and worsened in the placebo group, with more responders in the HCS group at 26 weeks. Neuropathic pain improved in the HCS group compared to placebo. There was a trend to benefit for lung function indices. Cluster analysis revealed changes in a number of cytokines in the HCS group compared to placebo, in parallel with the skin changes. In particular, α-MSH and ACTH were significantly increased in the HCS group leading use to hypothesise that improvement in MRSS may have been mediated through the melanocortin system.
Post-stroke cognitive impairment and dementia (PSCID) affects many survivors of large vessel cerebral ischemia. The molecular pathways underlying PSCID are poorly defined but may overlap with ...neurodegenerative pathophysiology. Specifically, synaptic dysfunction after stroke may be directly mediated by alterations in the levels of amyloid beta (Aβ), the peptide that accumulates in the brains of Alzheimer’s disease (AD) patients. In this study, we use the transient middle cerebral artery occlusion (MCAo) model in young adult mice to evaluate if a large vessel stroke increases brain soluble Aβ levels. We show that soluble Aβ40 and Aβ42 levels are increased in the ipsilateral hippocampus in MCAo mice 7 days after the injury. We also analyze the level and activity of β-site amyloid precursor protein cleaving enzyme 1 (BACE1), an enzyme that generates Aβ in the brain, and observe that BACE1 activity is increased in the ipsilateral hippocampus of the MCAo mice. Finally, we highlight that treatment of MCAo mice with a BACE1 inhibitor during the recovery period rescues stroke-induced deficits in hippocampal synaptic plasticity. These findings support a molecular pathway linking ischemia to alterations in BACE1-mediated production of Aβ, and encourage future studies that evaluate whether targeting BACE1 activity improves the cognitive deficits seen with PSCID.
Neonatal stroke is common and causes life‐long motor and cognitive sequelae. Because neonates with stroke are not diagnosed until days‐months after the injury, chronic targets for repair are needed. ...We evaluated oligodendrocyte maturity and myelination and assessed oligodendrocyte gene expression changes using single cell RNA sequencing (scRNA seq) at chronic timepoints in a mouse model of neonatal arterial ischemic stroke. Mice underwent 60 min of transient right middle cerebral artery occlusion (MCAO) on postnatal day 10 (p10) and received 5‐ethynyl‐2′‐deoxyuridine (EdU) on post‐MCAO days 3–7 to label dividing cells. Animals were sacrificed 14 and 28–30 days post‐MCAO for immunohistochemistry and electron microscopy. Oligodendrocytes were isolated from striatum 14 days post‐MCAO for scRNA seq and differential gene expression analysis. The density of Olig2+EdU+ cells was significantly increased in ipsilateral striatum 14 days post‐MCAO and the majority of oligodendrocytes were immature. Density of Olig2+EdU+ cells declined significantly between 14 and 28 days post‐MCAO without a concurrent increase in mature Olig2+EdU+ cells. By 28 days post‐MCAO there were significantly fewer myelinated axons in ipsilateral striatum. scRNA seq identified a cluster of “disease associated oligodendrocytes (DOLs)” specific to the ischemic striatum, with increased expression of MHC class I genes. Gene ontology analysis suggested decreased enrichment of pathways involved in myelin production in the reactive cluster. Oligodendrocytes proliferate 3–7 days post‐MCAO and persist at 14 days, but fail to mature by 28 days. MCAO induces a subset of oligodendrocytes with reactive phenotype, which may be a therapeutic target to promote white matter repair.
Main Points
Focal arterial ischemic stroke in full‐term equivalent neonatal mice alters proliferation and maturation of oligodendrocytes and myelination.
Single cell transcriptomics reveal a unique subset of oligodendrocytes with an immune phenotype that persist 2 weeks after ischemia.
The Earth's population is aging, and by 2050, one of six people will be 65 years or older. Therefore, proper treatment of injuries that disproportionately impact people of advanced age will be more ...important. Clinical studies reveal people 65 years or older account for 16.5% of all burn injuries and experience higher morbidity, including neurocognitive decline, and mortality that we and others believe are mediated, in part, by heightened intestinal permeability. Herein, we used our clinically relevant model of scald burn injury in young and aged mice to determine whether age and burn injury cooperate to induce heightened colonic damage, alterations to the fecal microbiome, and whether resultant changes in the microbiome correlate with neuroinflammation. We found that aged, burn-injured mice have an increase in colonic lymphoid aggregates, inflammation, and proinflammatory chemokine expression when compared with young groups and sham-injured aged mice. We then performed fecal microbiota sequencing and found a striking reduction in gut protective bacterial taxa, including Akkermansia , in the aged burn group compared with all other groups. This reduction correlated with an increase in serum fluorescein isothiocyanate-Dextran administered by gavage, indicating heightened intestinal permeability. Furthermore, loss of Akkermansia was highly correlated with increased messenger RNA expression of neuroinflammatory markers in the brain, including chemokine ligand 2, TNF-α, CXC motif ligand 1, and S100 calcium-binding protein A8. Finally, we discovered that postburn alterations in the microbiome correlated with measures of strength in all treatment groups, and those that performed better on the rotarod and hanging wire tests had higher abundance of Akkermansia than those that performed worse. Taken together, these findings indicate that loss of protective bacteria after burn injury in aged mice contributes to alterations in the colon, gut leakiness, neuroinflammation, and strength. Therefore, supplementation of protective bacteria, such as Akkermansia , after burn injury in aged patients may have therapeutic benefit.
Hyperimmune caprine serum (HICS) is a novel biological therapy with potential benefit for skin in established diffuse cutaneous systemic sclerosis. Here we report multiplex protein analysis of blood ...samples from a placebo-controlled phase II clinical trial and explore mechanisms of action and markers of response.
Patients were treated with HICS (n = 10) or placebo (n = 10) over 26 weeks, with follow-up open-label treatment to 52 weeks in 14 patients. Serum or plasma samples at baseline, 26 and 52 weeks were analysed using multiplex or individual immunoassays for 41 proteins. Patterns of change were analysed by clustering using Netwalker 1.0, Pearson coefficient and significance analysis of microarrays (SAM) correction.
Cluster analysis, SAM multiplex testing and paired comparison of individual analytes identified proteins that were upregulated or downregulated during treatment with HICS. There was upregulation of the hypothalamo-pituitary-adrenal axis after HICS treatment evidenced by increases in α-MSH and ACTH in cases treated with HICS. Interestingly, significant increase in PIIINP was associated with HICS treatment and improved MRSS suggesting that this may be a marker of extracellular matrix turnover. Other relevant factors reduced in HICS-treated patients compared with controls, although not reaching statistical significance included COMP, CCL2, IL6, TIMP2, Fractalkine and TGFβ1 levels.
Our results suggest mechanisms of action for HICS, including upregulation of α-MSH, that has been shown to be anti-fibrotic in preclinical models, and possible markers to be included in future trials targeting skin in diffuse cutaneous systemic sclerosis.
Eudract, No. 2007-003122-24. ClinTrials.gov, No. NCT00769028 . Registered 7 October 2008.
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