Degree of COVID-19 severity and mortality in stroke: correlation of clinical and laboratory parameters.

BACKGROUND: Stroke is one of the neurological manifestations of COVID-19, leading to a significant risk of morbidity and mortality. Clinical manifestations and laboratory parameters were investigated to determine mortality predictors in this case. METHOD: The case control study was conducted at Dr. Sardjito General Hospital,Yogyakarta, Indonesia, with data collected between July 2020 and August 2021. All recorded clinical and laboratory data from acute stroke patients with confirmed COVID-19 were collected. Baseline characteristics, bivariate, and multivariate analyses were assessed to determine significant predictors for mortality. RESULT: This study involved 72 subjects with COVID-19 and stroke. The majority experienced ischemic stroke, with hypertension as the most prevalent comorbidity. Notably, 45.8% of subjects (p < 0.05) loss of consciousness and 72.2% of exhibited motor deficits (p < 0.05). Severe degree of COVID-19 was observed in 52.8% of patients, with respiratory distress and death rates of 56.9% and 58.3%. Comparison of surviving and deceased groups highlighted significant differences in various clinical and laboratory characteristics differences. Hazard ratio (HR) analysis identified loss of consciousness (HR = 2.68; p = 0.01), motor deficit (HR = 2.34; p = 0.03), respiratory distress (HR = 81.51; p < 0.001), and monocyte count (HR:1.002; p = 0.04) as significant predictors of mortality. CONCLUSION: Mortality in COVID-19 patients with stroke was significantly associated with loss of consciousness, motor deficit, respiratory distress, and raised monocyte count. The risk of mortality is heightened when multiple factors coexist.

Brain-derived neurotrophic factor interplay with oxidative stress: neuropathology approach in potential biomarker of Alzheimer's disease.

The aging population poses a serious challenge concerning an increased prevalence of Alzheimer's disease (AD) and its impact on global burden, morbidity, and mortality. Oxidative stress, as a molecular hallmark that causes susceptibility in AD, interplays to other AD-related neuropathology cascades and decreases the expression of central and circulation brain-derived neurotrophic factor (BDNF), an essential neurotrophin that serves as nerve development and survival, and synaptic plasticity in AD. By its significant correlation with the molecular and clinical progression of AD, BDNF can potentially be used as an objectively accurate biomarker for AD diagnosis and progressivity follow-up in future clinical practice. This comprehensive review highlights the oxidative stress interplay with BDNF in AD neuropathology and its potential use as an AD biomarker.

O envelhecimento da população representa um sério desafio no que diz respeito ao aumento da prevalência da doença de Alzheimer (DA) e o seu impacto na carga, morbidade e mortalidade globais. O estresse oxidativo, como uma marca molecular que causa suscetibilidade na DA, interage com outras cascatas de neuropatologia relacionadas à DA e diminui a expressão do fator neurotrófico encefálico (brain-derived neurotrophic factor ­ BDNF), uma neurotrofina essencial que serve como desenvolvimento e sobrevivência nervosa, e plasticidade sináptica na DA. Pela sua correlação significativa com a progressão molecular e clínica da DA, o BDNF pode potencialmente ser usado como um biomarcador objetivamente preciso para o diagnóstico da DA e acompanhamento da progressividade na prática clínica futura. Esta revisão abrangente destacou a interação do estresse oxidativo com o BDNF na neuropatologia da DA e seu uso potencial como biomarcador da DA.

Brain-derived neurotrophic factor interplay with oxidative stress: neuropathology approach in potential biomarker of Alzheimer's disease / Interação do fator neurotrófico encefálico com o estresse oxidativo: abordagem neuropatológica a um potencial biomarcador da doença de Alzheimer

ABSTRACT The aging population poses a serious challenge concerning an increased prevalence of Alzheimer's disease (AD) and its impact on global burden, morbidity, and mortality. Oxidative stress, as a molecular hallmark that causes susceptibility in AD, interplays to other AD-related neuropathology cascades and decreases the expression of central and circulation brain-derived neurotrophic factor (BDNF), an essential neurotrophin that serves as nerve development and survival, and synaptic plasticity in AD. By its significant correlation with the molecular and clinical progression of AD, BDNF can potentially be used as an objectively accurate biomarker for AD diagnosis and progressivity follow-up in future clinical practice. This comprehensive review highlights the oxidative stress interplay with BDNF in AD neuropathology and its potential use as an AD biomarker.

RESUMO O envelhecimento da população representa um sério desafio no que diz respeito ao aumento da prevalência da doença de Alzheimer (DA) e o seu impacto na carga, morbidade e mortalidade globais. O estresse oxidativo, como uma marca molecular que causa suscetibilidade na DA, interage com outras cascatas de neuropatologia relacionadas à DA e diminui a expressão do fator neurotrófico encefálico (brain-derived neurotrophic factor - BDNF), uma neurotrofina essencial que serve como desenvolvimento e sobrevivência nervosa, e plasticidade sináptica na DA. Pela sua correlação significativa com a progressão molecular e clínica da DA, o BDNF pode potencialmente ser usado como um biomarcador objetivamente preciso para o diagnóstico da DA e acompanhamento da progressividade na prática clínica futura. Esta revisão abrangente destacou a interação do estresse oxidativo com o BDNF na neuropatologia da DA e seu uso potencial como biomarcador da DA.

Folic Acid Treatment for Patients with Vascular Cognitive Impairment: A Systematic Review and Meta-Analysis.

BACKGROUND: As the life expectancy of elderly people has drastically increased, the incidence of cardiovascular and cerebrovascular diseases in this population has proportionally grown. Vascular cognitive impairment (VCI) refers to all forms of cognitive disorder associated with cerebrovascular disease. Homocysteine has recently been recognized as a contributor to the pathomechanisms involved in cognitive impairment. B vitamins, such as folic acid, are known to be effective in lowering homocysteine levels. AIM OF THE STUDY: To evaluate the efficacy of folic acid in patients with VCI. METHODS: We conducted a systematic review and meta-analysis of research on folic acid treatments for VCI. Only randomized controlled trials studies that compared the efficacy of folic acid to placebo or other interventions were considered, irrespective of publication status, year of publication, and languages. Two independent reviewers searched the Medline via Ovid, EMBASE and Cochrane Central Register of Controlled Trials (Central) journal databases up to July 2021 and independently appraised the included studies. We used mean difference outcome with 95% confidence intervals (CI) to calculate the change of Mini-Mental State Examination (MMSE), cognitive function domain, and concentration of homocysteine. RESULTS: We found three studies comparing folic acid with placebo and one study comparing folic acid with other interventions. There is only slight evidence that the MMSE score in patients who received Folic Acid increased 0.3 point higher compared to the placebo group after 24 months (95% CI:-0.12-0.37; p=0.31). There is very strong evidence that the concentration of Homocysteine in the Folic Acid group became 6.16 µmol/L lower compared to the placebo group after 6 months (95% CI:2.32-8.21 lower; p<0.001). CONCLUSIONS: Our review shows the effectiveness of folic acid in lowering plasma homocysteine concentration after 6 months period compared to placebo. However, this effect is not accompanied by improvement in cognitive function.

Adenosine 2A receptor inhibition protects phrenic motor neurons from cell death induced by protein synthesis inhibition.

Respiratory motor neuron survival is critical for maintenance of adequate ventilation and airway clearance, preventing dependence to mechanical ventilation and respiratory tract infections. Phrenic motor neurons are highly vulnerable in rodent models of motor neuron disease versus accessory inspiratory motor pools (e.g. intercostals, scalenus). Thus, strategies that promote phrenic motor neuron survival when faced with disease and/or toxic insults are needed to help preserve breathing ability, airway defense and ventilator independence. Adenosine 2A receptors (A2A) are emerging as a potential target to promote neuroprotection, although their activation can have both beneficial and pathogenic effects. Since the role of A2A receptors in the phrenic motor neuron survival/death is not known, we tested the hypothesis that A2A receptor antagonism promotes phrenic motor neuron survival and preserves diaphragm function when faced with toxic, neurodegenerative insults that lead to phrenic motor neuron death. We utilized a novel neurotoxic model of respiratory motor neuron death recently developed in our laboratory: intrapleural injections of cholera toxin B subunit (CtB) conjugated to the ribosomal toxin, saporin (CtB-Saporin). We demonstrate that intrapleural CtB-Saporin causes: 1) profound phrenic motor neuron death (~5% survival); 2) ~7-fold increase in phrenic motor neuron A2A receptor expression prior to cell death; and 3) diaphragm muscle paralysis (inactive in most rats; ~7% residual diaphragm EMG amplitude during room air breathing). The A2A receptor antagonist istradefylline given after CtB-Saporin: 1) reduced phrenic motor neuron death (~20% survival) and 2) preserved diaphragm EMG activity (~46%). Thus, A2A receptors contribute to neurotoxic phrenic motor neuron death, an effect mitigated by A2A receptor antagonism.

Compensatory plasticity in diaphragm and intercostal muscle utilization in a rat model of ALS.

In SOD1G93A transgenic rat model of ALS, breathing capacity is preserved until late in disease progression despite profound respiratory motor neuron (MN) cell death. To explore mechanisms preserving breathing capacity, we assessed inspiratory EMG activity in diaphragm and external intercostal T2 (EIC2) and T5 (EIC5) muscles in anesthetized SOD1G93A rats at disease end-stage (20% decrease in body mass). We hypothesized that despite significant phrenic motor neuron loss and decreased phrenic nerve activity, diaphragm electrical activity and trans-diaphragmatic pressure (Pdi) are maintained to sustain ventilation. We alternatively hypothesized that EIC activity is enhanced, compensating for impaired diaphragm function. Diaphragm, EIC2 and EIC5 muscle EMGs and Pdi were measured in urethane-anesthetized, spontaneously breathing female SOD1G93A rats versus wild-type littermates during normoxia (arterial PO2 ~90mmHg, PCO2 ~45mmHg), maximal chemoreceptor stimulation (MCS: 10.5% O2/7% CO2), spontaneous augmented breaths and sustained tracheal occlusion. Phrenic MNs were counted in C3-5; T2 and T5 ventrolateral MNs were counted. In end-stage SOD1G93A rats, 29% of phrenic MNs survived (vs. wild-type), yet integrated diaphragm EMG amplitude was normal. Nevertheless, maximal Pdi decreased ~30% vs. wild type (p<0.01) and increased esophageal to gastric pressure ratio (p<0.05), consistent with persistent diaphragm weakness. Despite major T2 and T5 MN death, integrated EIC2 (100% greater than wild type) and EIC5 (300%) EMG amplitudes were increased in mutant rats during normoxia (p<0.01), possibly compensating for decreased Pdi. Thus, despite significant phrenic MN loss, diaphragm EMG activity is maintained; in contrast, Pdi was not, suggesting diaphragm dysfunction. Presumably, increased EIC EMG activity compensated for persistent diaphragm weakness. These adjustments contribute to remarkable preservation of breathing ability despite major respiratory motor neuron death and diaphragm dysfunction.

Mechanisms of Enhanced Phrenic Long-Term Facilitation in SOD1G93A Rats.

Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron disease, causing muscle paralysis and death from respiratory failure. Effective means to preserve/restore ventilation are necessary to increase the quality and duration of life in ALS patients. At disease end-stage in a rat ALS model (SOD1G93A ), acute intermittent hypoxia (AIH) restores phrenic nerve activity to normal levels via enhanced phrenic long-term facilitation (pLTF). Mechanisms enhancing pLTF in end-stage SOD1G93A rats are not known. Moderate AIH-induced pLTF is normally elicited via cellular mechanisms that require the following: Gq-protein-coupled 5-HT2 receptor activation, new BDNF synthesis, and MEK/ERK signaling (the Q pathway). In contrast, severe AIH elicits pLTF via a distinct mechanism that requires the following: Gs-protein-coupled adenosine 2A receptor activation, new TrkB synthesis, and PI3K/Akt signaling (the S pathway). In end-stage male SOD1G93A rats and wild-type littermates, we investigated relative Q versus S pathway contributions to enhanced pLTF via intrathecal (C4) delivery of small interfering RNAs targeting BDNF or TrkB mRNA, and MEK/ERK (U0126) or PI3 kinase/Akt (PI828) inhibitors. In anesthetized, paralyzed and ventilated rats, moderate AIH-induced pLTF was abolished by siBDNF and UO126, but not siTrkB or PI828, demonstrating that enhanced pLTF occurs via the Q pathway. Although phrenic motor neuron numbers were decreased in end-stage SOD1G93A rats (∼30% survival; p < 0.001), BDNF and phosphorylated ERK expression were increased in spared phrenic motor neurons (p < 0.05), consistent with increased Q-pathway contributions to pLTF. Our results increase understanding of respiratory plasticity and its potential to preserve/restore breathing capacity in ALS.SIGNIFICANCE STATEMENT Since neuromuscular disorders, such as amyotrophic lateral sclerosis (ALS), end life via respiratory failure, the ability to harness respiratory motor plasticity to improve breathing capacity could increase the quality and duration of life. In a rat ALS model (SOD1G93A ) we previously demonstrated that spinal respiratory motor plasticity elicited by acute intermittent hypoxia is enhanced at disease end-stage, suggesting greater potential to preserve/restore breathing capacity. Here we demonstrate that enhanced intermittent hypoxia-induced phrenic motor plasticity results from amplification of normal cellular mechanisms versus addition/substitution of alternative mechanisms. Greater understanding of mechanisms underlying phrenic motor plasticity in ALS may guide development of new therapies to preserve and/or restore breathing in ALS patients.

Considerations for the Optimization of Induced White Matter Injury Preclinical Models.

White matter (WM) injury in relation to acute neurologic conditions, especially stroke, has remained obscure until recently. Current advances in imaging technologies in the field of stroke have confirmed that WM injury plays an important role in the prognosis of stroke and suggest that WM protection is essential for functional recovery and post-stroke rehabilitation. However, due to the lack of a reproducible animal model of WM injury, the pathophysiology and mechanisms of this injury are not well studied. Moreover, producing selective WM injury in animals, especially in rodents, has proven to be challenging. Problems associated with inducing selective WM ischemic injury in the rodent derive from differences in the architecture of the brain, most particularly, the ratio of WM to gray matter in rodents compared to humans, the agents used to induce the injury, and the location of the injury. Aging, gender differences, and comorbidities further add to this complexity. This review provides a brief account of the techniques commonly used to induce general WM injury in animal models (stroke and non-stroke related) and highlights relevance, optimization issues, and translational potentials associated with this particular form of injury.

Acute intermittent hypoxia induced phrenic long-term facilitation despite increased SOD1 expression in a rat model of ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease characterized by motor neuron death. Since most ALS patients succumb to ventilatory failure from loss of respiratory motor neurons, any effective ALS treatment must preserve and/or restore breathing capacity. In rats over-expressing mutated super-oxide dismutase-1 (SOD1(G93A)), the capacity to increase phrenic motor output is decreased at disease end-stage, suggesting imminent ventilatory failure. Acute intermittent hypoxia (AIH) induces phrenic long-term facilitation (pLTF), a form of spinal respiratory motor plasticity with potential to restore phrenic motor output in clinical disorders that compromise breathing. Since pLTF requires NADPH oxidase activity and reactive oxygen species (ROS) formation, it is blocked by NADPH oxidase inhibition and SOD mimetics in normal rats. Thus, we hypothesized that SOD1(G93A) (mutant; MT) rats do not express AIH-induced pLTF due to over-expression of active mutant superoxide dismutase-1. AIH-induced pLTF and hypoglossal (XII) LTF were assessed in young, pre-symptomatic and end-stage anesthetized MT rats and age-matched wild-type littermates. Contrary to predictions, pLTF and XII LTF were observed in MT rats at all ages; at end-stage, pLTF was actually enhanced. SOD1 levels were elevated in young and pre-symptomatic MT rats, yet superoxide accumulation in putative phrenic motor neurons (assessed with dihydroethidium) was unchanged; however, superoxide accumulation significantly decreased at end-stage. Thus, compensatory mechanisms appear to maintain ROS homoeostasis until late in disease progression, preserving AIH-induced respiratory plasticity. Following intrathecal injections of an NADPH oxidase inhibitor (apocynin; 600 µM; 12 µL), pLTF was abolished in pre-symptomatic, but not end-stage MT rats, demonstrating that pLTF is NADPH oxidase dependent in pre-symptomatic, but NADPH oxidase independent in end-stage MT rats. Mechanisms preserving/enhancing the capacity for pLTF in MT rats are not known.

Optimization of a Clinically Relevant Model of White Matter Stroke in Mice: Histological and Functional Evidences.

BACKGROUND AND PURPOSE: White matter (WM) injury during stroke increases the risk of disability and gloomy prognosis of post-stroke rehabilitation. However, modeling of WM loss in rodents has proven to be challenging. METHODS: We report improved WM injury models in male C57BL/6 mice. Mice were given either endothelin-1 (ET-1) or L-N5-(1-iminoethyl)ornitine (L-NIO) into the periventricular white matter (PVWM), in the corpus callosum (CC), or in the posterior limb of internal capsule (PLIC). Anatomical and functional outcomes were quantified on day 7 post injection. RESULTS: Injection of ET-1 or L-NIO caused a small focal lesion in the injection site in the PVWM. No significant motor function deficits were observed in the PVWM lesion model. We next targeted the PLIC by using single or double injections of L-NIO and found that this strategy induced small focal infarction. Interestingly, injection of L-NIO in the PLIC also resulted in gliosis, and significant motor function deficits. CONCLUSIONS: By employing different agents, doses, and locations, this study shows the feasibility of inducing brain WM injury accompanied with functional deficits in mice. Selective targeting of the injury location, behavioral testing, and the agents chosen to induce WM injury are all keys to successfully develop a mouse model and subsequent testing of therapeutic interventions against WM injury.

Treatment of stroke related refractory brain edema using mixed vasopressin antagonism: a case report and review of the literature.

BACKGROUND: Elevated intracranial pressure from cerebral edema is the major cause of early mortality in acute stroke. Current treatment strategies to limit cerebral edema are not particularly effective. Some novel anti-edema measures have shown promising early findings in experimental stroke models. Vasopressin antagonism in stroke is one such target which has shown some encouraging preliminary results. The aim of this report is to highlight the potential use of vasopressin antagonism to limit cerebral edema in patients after acute stroke. CASE PRESENTATION: A 57-year-old Caucasian man with new onset diplopia was diagnosed with vertebral artery aneurysm extending into the basilar circulation. He underwent successful elective vertebral artery angioplasty and coiling of the aneurysm. In the immediate post-operative period there was a decline in his neurological status and brain imaging revealed new midbrain and thalamic hemorrhage with surrounding significant brain edema. Treatment with conventional anti-edema therapy was initiated with no significant clinical response after which conivaptan; a mixed vasopressin antagonist was started. Clinical and radiological evaluation following drug administration showed rapid clinical improvement without identification of significant adverse effects. CONCLUSIONS: The authors have successfully demonstrated the safety and efficacy of using mixed vasopressin antagonist in treatment of stroke related brain edema, thereby showing its promise as an alternative anti-edema agent. Preliminary findings from this study suggest mixed vasopressin antagonism may have significant utility in the management of cerebral edema arising from cerebrovascular accident. Larger prospective studies are warranted to explore the role of conivaptan in the treatment of brain edema and neuroprotection.

Role of vasopressin and its antagonism in stroke related edema.

Although many approaches have been tried in the attempt to reduce the devastating impact of stroke, tissue plasminogen activator for thromboembolic stroke is the only proved, effective acute stroke treatment to date. Vasopressin, an acute-phase reactant, is released after brain injury and is partially responsible for the subsequent inflammatory response via activation of divergent pathways. Recently there has been increasing interest in vasopressin because it is implicated in inflammation, cerebral edema, increased intracerebral pressure, and cerebral ion and neurotransmitter dysfunctions after cerebral ischemia. Additionally, copeptin, a byproduct of vasopressin production, may serve as a promising independent marker of tissue damage and prognosis after stroke, thereby corroborating the role of vasopressin in acute brain injury. Thus, vasopressin antagonists have a potential role in early stroke intervention, an effect thought to be mediated via interactions with aquaporin receptors, specifically aquaporin-4. Despite some ambiguity, vasopressin V1a receptor antagonism has been consistently associated with attenuated secondary brain injury and edema in experimental stroke models. The role of the vasopressin V2 receptor remains unclear, but perhaps it is involved in a positive feedback loop for vasopressin expression. Despite the encouraging initial findings we report here, future research is required to characterize further the utility of vasopressin antagonists in treatment of stroke.

Intermittent hypoxia and stem cell implants preserve breathing capacity in a rodent model of amyotrophic lateral sclerosis.

RATIONALE: Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease causing paralysis and death from respiratory failure. Strategies to preserve and/or restore respiratory function are critical for successful treatment. Although breathing capacity is maintained until late in disease progression in rodent models of familial ALS (SOD1(G93A) rats and mice), reduced numbers of phrenic motor neurons and decreased phrenic nerve activity are observed. Decreased phrenic motor output suggests imminent respiratory failure. OBJECTIVES: To preserve or restore phrenic nerve activity in SOD1(G93A) rats at disease end stage. METHODS: SOD1(G93A) rats were injected with human neural progenitor cells (hNPCs) bracketing the phrenic motor nucleus before disease onset, or exposed to acute intermittent hypoxia (AIH) at disease end stage. MEASUREMENTS AND MAIN RESULTS: The capacity to generate phrenic motor output in anesthetized rats at disease end stage was: (1) transiently restored by a single presentation of AIH; and (2) preserved ipsilateral to hNPC transplants made before disease onset. hNPC transplants improved ipsilateral phrenic motor neuron survival. CONCLUSIONS: AIH-induced respiratory plasticity and stem cell therapy have complementary translational potential to treat breathing deficits in patients with ALS.

Repetitive acute intermittent hypoxia increases expression of proteins associated with plasticity in the phrenic motor nucleus.

Acute intermittent hypoxia (AIH) initiates plasticity in respiratory motor control, including phrenic long term facilitation (pLTF). Since pLTF is enhanced by preconditioning with repetitive exposure to AIH (rAIH), we hypothesized that a rAIH protocol consisting of 3 AIH exposures per week for 10 weeks (3×wAIH; AIH: 10, 5-min episodes of 10.5% O(2); 5-min normoxic intervals) would enhance expression of molecules that play key roles in pLTF within the phrenic motor nucleus. Immunohistochemical analyses revealed that 3×wAIH for 10 weeks increased serotonin terminal density in the C4 phrenic motor nucleus and serotonin 2A (5-HT(2A)) receptor expression in presumptive phrenic motor neurons. Immunoreactive brain derived neurotrophic factor (BDNF) and its high affinity receptor (TrkB) also increased following 3×wAIH. 3×wAIH also increased expression of another hypoxia-sensitive growth factor known to elicit phrenic motor facilitation, vascular endothelial growth factor (VEGF), and its receptor (VEGFR-2). Kinases "downstream" from TrkB and VEGFR-2 were up-regulated in or near presumptive phrenic motor neurons, including phosphorylated extracellular-signal regulated kinase (p-ERK) and protein kinase B (p-AKT). Thus, 3×wAIH up-regulates neurochemicals known to be associated with phrenic motor plasticity. Since 3×wAIH upregulates pro-plasticity molecules without evidence for CNS pathology, it may be a useful therapeutic tool in treating disorders that cause respiratory insufficiency, such as spinal injury or motor neuron disease.

Cervical spinal erythropoietin induces phrenic motor facilitation via extracellular signal-regulated protein kinase and Akt signaling.

Erythropoietin (EPO) is typically known for its role in erythropoiesis but is also a potent neurotrophic/neuroprotective factor for spinal motor neurons. Another trophic factor regulated by hypoxia-inducible factor-1, vascular endothelial growth factor (VEGF), signals via ERK and Akt activation to elicit long-lasting phrenic motor facilitation (pMF). Because EPO also signals via ERK and Akt activation, we tested the hypothesis that EPO elicits similar pMF. Using retrograde labeling and immunohistochemical techniques, we demonstrate in adult, male, Sprague Dawley rats that EPO and its receptor, EPO-R, are expressed in identified phrenic motor neurons. Intrathecal EPO at C4 elicits long-lasting pMF; integrated phrenic nerve burst amplitude increased >90 min after injection (63 ± 12% baseline 90 min after injection; p < 0.001). EPO increased phosphorylation (and presumed activation) of ERK (1.6-fold vs controls; p < 0.05) in phrenic motor neurons; EPO also increased pAkt (1.6-fold vs controls; p < 0.05). EPO-induced pMF was abolished by the MEK/ERK inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene] and the phosphatidylinositol 3-kinase/Akt inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one], demonstrating that ERK MAP kinases and Akt are both required for EPO-induced pMF. Pretreatment with U0126 and LY294002 decreased both pERK and pAkt in phrenic motor neurons (p < 0.05), indicating a complex interaction between these kinases. We conclude that EPO elicits spinal plasticity in respiratory motor control. Because EPO expression is hypoxia sensitive, it may play a role in respiratory plasticity in conditions of prolonged or recurrent low oxygen.

Repetitive intermittent hypoxia induces respiratory and somatic motor recovery after chronic cervical spinal injury.

Spinal injury disrupts connections between the brain and spinal cord, causing life-long paralysis. Most spinal injuries are incomplete, leaving spared neural pathways to motor neurons that initiate and coordinate movement. One therapeutic strategy to induce functional motor recovery is to harness plasticity in these spared neural pathways. Chronic intermittent hypoxia (CIH) (72 episodes per night, 7 nights) increases synaptic strength in crossed spinal synaptic pathways to phrenic motoneurons below a C2 spinal hemisection. However, CIH also causes morbidity (e.g., high blood pressure, hippocampal apoptosis), rendering it unsuitable as a therapeutic approach to chronic spinal injury. Less severe protocols of repetitive acute intermittent hypoxia may elicit plasticity without associated morbidity. Here we demonstrate that daily acute intermittent hypoxia (dAIH; 10 episodes per day, 7 d) induces motor plasticity in respiratory and nonrespiratory motor behaviors without evidence for associated morbidity. dAIH induces plasticity in spared, spinal pathways to respiratory and nonrespiratory motor neurons, improving respiratory and nonrespiratory (forelimb) motor function in rats with chronic cervical injuries. Functional improvements were persistent and were mirrored by neurochemical changes in proteins that contribute to respiratory motor plasticity after intermittent hypoxia (BDNF and TrkB) within both respiratory and nonrespiratory motor nuclei. Collectively, these studies demonstrate that repetitive acute intermittent hypoxia may be an effective and non-invasive means of improving function in multiple motor systems after chronic spinal injury.

Spinal vascular endothelial growth factor induces phrenic motor facilitation via extracellular signal-regulated kinase and Akt signaling.

Although vascular endothelial growth factor (VEGFA-165) is primarily known for its role in angiogenesis, it also plays important neurotrophic and neuroprotective roles for spinal motor neurons. VEGFA-165 signals by activating its receptor tyrosine kinase VEGF receptor-2 (VEGFR-2). Because another growth/trophic factor that signals via a receptor tyrosine kinase (brain derived neurotrophic factor) elicits a long-lasting facilitation of respiratory motor activity in the phrenic nerve, we tested the hypothesis that VEGFA-165 elicits similar phrenic motor facilitation (pMF). Using immunohistochemistry and retrograde labeling techniques, we demonstrate that VEGFA-165 and VEGFR-2 are expressed in identified phrenic motor neurons. Furthermore, intrathecal VEGFA-165 administration at C4 elicits long-lasting pMF; intraspinal VEGFA-165 increased integrated phrenic nerve burst amplitude for at least 90 min after injection (53.1 ± 5.0% at 90 min; p < 0.001). Intrathecal VEGFA-165 increased phosphorylation (and presumed activation) of signaling molecules downstream from VEGFR-2 within the phrenic motor nucleus, including ERK (1.53 ± 0.13 vs 1.0 ± 0.05 arbitrary units in control rats; p < 0.05) and Akt (2.16 ± 0.41 vs 1.0 ± 0.41 arbitrary units in control rats; p < 0.05). VEGF-induced pMF was attenuated by the MEK/ERK inhibitor U0126 [1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene] and was abolished by the phosphotidinositol 3 kinase/Akt inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride], demonstrating that ERK mitogen-activated protein kinases and Akt are both required for full expression of VEGF-induced pMF. This is the first report that VEGFA-165 elicits plasticity in any motor system. Furthermore, because VEGFA-165 expression is hypoxia sensitive, it may play a role in respiratory plasticity after prolonged exposures to low oxygen.

Spinal plasticity following intermittent hypoxia: implications for spinal injury.

Plasticity is a fundamental property of the neural system controlling breathing. One frequently studied model of respiratory plasticity is long-term facilitation of phrenic motor output (pLTF) following acute intermittent hypoxia (AIH). pLTF arises from spinal plasticity, increasing respiratory motor output through a mechanism that requires new synthesis of brain-derived neurotrophic factor, activation of its high-affinity receptor, tropomyosin-related kinase B, and extracellular-related kinase mitogen-activated protein kinase signaling in or near phrenic motor neurons. Because intermittent hypoxia induces spinal plasticity, we are exploring the potential to harness repetitive AIH as a means of inducing functional recovery in conditions causing respiratory insufficiency, such as cervical spinal injury. Because repetitive AIH induces phenotypic plasticity in respiratory motor neurons, it may restore respiratory motor function in patients with incomplete spinal injury.

Okadaic acid-sensitive protein phosphatases constrain phrenic long-term facilitation after sustained hypoxia.

Phrenic long-term facilitation (pLTF) is a serotonin-dependent form of pattern-sensitive respiratory plasticity induced by intermittent hypoxia (IH), but not sustained hypoxia (SH). The mechanism(s) underlying pLTF pattern sensitivity are unknown. SH and IH may differentially regulate serine/threonine protein phosphatase activity, thereby inhibiting relevant protein phosphatases uniquely during IH and conferring pattern sensitivity to pLTF. We hypothesized that spinal protein phosphatase inhibition would relieve this braking action of protein phosphatases, thereby revealing pLTF after SH. Anesthetized rats received intrathecal (C4) okadaic acid (25 nm) before SH (25 min, 11% O(2)). Unlike (vehicle) control rats, SH induced a significant pLTF in okadaic acid-treated rats that was indistinguishable from rats exposed to IH (three 5 min episodes, 11% O(2)). IH and SH with okadaic acid may elicit pLTF by similar, serotonin-dependent mechanisms, because intravenous methysergide blocks pLTF in rats receiving IH or okadaic acid plus SH. Okadaic acid did not alter IH-induced pLTF. In summary, pattern sensitivity in pLTF may reflect differential regulation of okadaic acid-sensitive serine/threonine phosphatases; presumably, these phosphatases are less active during/after IH versus SH. The specific okadaic acid-sensitive phosphatase(s) constraining pLTF and their spatiotemporal dynamics during and/or after IH and SH remain to be determined.

Spinal adenosine A2a receptor activation elicits long-lasting phrenic motor facilitation.

Acute intermittent hypoxia elicits a form of spinal, brain-derived neurotrophic factor (BDNF)-dependent respiratory plasticity known as phrenic long-term facilitation. Ligands that activate G(s)-protein-coupled receptors, such as the adenosine 2a receptor, mimic the effects of neurotrophins in vitro by transactivating their high-affinity receptor tyrosine kinases, the Trk receptors. Thus, we hypothesized that A2a receptor agonists would elicit phrenic long-term facilitation by mimicking the effects of BDNF on TrkB receptors. Here we demonstrate that spinal A2a receptor agonists transactivate TrkB receptors in the rat cervical spinal cord near phrenic motoneurons, thus inducing long-lasting (hours) phrenic motor facilitation. A2a receptor activation increased phosphorylation and new synthesis of an immature TrkB protein, induced TrkB signaling through Akt, and strengthened synaptic pathways to phrenic motoneurons. RNA interference targeting TrkB mRNA demonstrated that new TrkB protein synthesis is necessary for A2a-induced phrenic motor facilitation. A2a receptor activation also increased breathing in unanesthetized rats, and improved breathing in rats with cervical spinal injuries. Thus, small, highly permeable drugs (such as adenosine receptor agonists) that transactivate TrkB receptors may provide an effective therapeutic strategy in the treatment of patients with ventilatory control disorders, such as obstructive sleep apnea, or respiratory insufficiency after spinal injury or during neurodegenerative diseases.