Treatment of vascular dementia in female rats with AV-001, an Angiopoietin-1 mimetic peptide, improves cognitive function.

Background: Vascular dementia (VaD) is a complex neurodegenerative disorder. We previously found that treatment of VaD in middle-aged male rats subjected to multiple microinfarction (MMI) with AV-001, a Tie2 receptor agonist, significantly improves cognitive function. Age and sex affect the development and response of VaD to therapeutic intervention. Thus, the present study investigated the therapeutic effect of AV-001 on VaD in aged female rats subjected to MMI. Methods: Female 18-month-old Wistar rats were subjected to MMI by injecting either 1,000 (low dose, LD-MMI) or 6,000 (high dose, HD-MMI) cholesterol crystals of size 70-100 µm into the right internal carotid artery. AV-001 (1 µg/Kg, i.p.) was administered once daily after MMI for 1 month, with treatment initiated 1 day after MMI. A battery of behavioral tests to examine sensorimotor and cognitive functions was performed at 21-28 days after MMI. All rats were sacrificed at 1 month after MMI. Results: Aged female rats subjected to LD-MMI exhibit severe neurological deficits, memory impairment, and significant white matter (WM) and oligodendrogenesis injury in the corpus callosum compared with control rats. HD-MMI in aged female rats induces significant anxiety- and depression-like behaviors, which were not detected in LD-MMI aged female rats. Also, HD-MMI induces significantly increased WM injury compared to LD-MMI. AV-001 treatment of LD-MMI and HD-MMI increases oligodendrogenesis, myelin and axon density in the corpus callosum and striatal WM bundles, promotes WM integrity and attenuates neurological and cognitive deficits. Additionally, both LD-MMI and HD-MMI rats exhibit a significant increase, while AV-001 significantly decreases the levels of inflammatory factors in the cerebrospinal fluid (CSF). Conclusion: MMI reduces oligodendrogenesis, and induces demyelination, axonal injury and WM injury, and causes memory impairment, while HD-MMI induces increased WM injury and further depression-like behaviors compared to LD-MMI rats. AV-001 has a therapeutic effect on aged female rats with MMI by reducing WM damage and improving neuro-cognitive outcomes.

Early therapeutic effects of an Angiopoietin-1 mimetic peptide in middle-aged rats with vascular dementia.

Background: Vascular Dementia (VaD) refers to dementia caused by cerebrovascular disease and/or reduced blood flow to the brain and is the second most common form of dementia after Alzheimer's disease. We previously found that in middle-aged rats subjected to a multiple microinfarction (MMI) model of VaD, treatment with AV-001, a Tie2 receptor agonist, significantly improves short-term memory, long-term memory, as well as improves preference for social novelty compared to control MMI rats. In this study, we tested the early therapeutic effects of AV-001 on inflammation and glymphatic function in rats subjected to VaD. Methods: Male, middle-aged Wistar rats (10-12 m), subjected to MMI, were randomly assigned to MMI and MMI + AV-001 treatment groups. A sham group was included as reference group. MMI was induced by injecting 800 ± 200, 70-100 µm sized, cholesterol crystals into the internal carotid artery. Animals were treated with AV-001 (1 µg/Kg, i.p.) once daily starting at 24 h after MMI. At 14 days after MMI, inflammatory factor expression was evaluated in cerebrospinal fluid (CSF) and brain. Immunostaining was used to evaluate white matter integrity, perivascular space (PVS) and perivascular Aquaporin-4 (AQP4) expression in the brain. An additional set of rats were prepared to test glymphatic function. At 14 days after MMI, 50 µL of 1% Tetramethylrhodamine (3 kD) and FITC conjugated dextran (500 kD) at 1:1 ratio were injected into the CSF. Rats (4-6/group/time point) were sacrificed at 30 min, 3 h, and 6 h from the start of tracer infusion, and brain coronal sections were imaged using a Laser scanning confocal microscope to evaluate tracer intensities in the brain. Result: Treatment of MMI with AV-001 significantly improves white matter integrity in the corpus callosum at 14 days after MMI. MMI induces significant dilation of the PVS, reduces AQP4 expression and impairs glymphatic function compared to Sham rats. AV-001 treatment significantly reduces PVS, increases perivascular AQP4 expression and improves glymphatic function compared to MMI rats. MMI significantly increases, while AV-001 significantly decreases the expression of inflammatory factors (tumor necrosis factor-α (TNF-α), chemokine ligand 9) and anti-angiogenic factors (endostatin, plasminogen activator inhibitor-1, P-selectin) in CSF. MMI significantly increases, while AV-001 significantly reduces brain tissue expression of endostatin, thrombin, TNF-α, PAI-1, CXCL9, and interleukin-6 (IL-6). Conclusion: AV-001 treatment of MMI significantly reduces PVS dilation and increases perivascular AQP4 expression which may contribute to improved glymphatic function compared to MMI rats. AV-001 treatment significantly reduces inflammatory factor expression in the CSF and brain which may contribute to AV-001 treatment induced improvement in white matter integrity and cognitive function.

Therapeutic effects of CD133 + Exosomes on liver function after stroke in type 2 diabetic mice.

Background and purpose: Non-alcoholic fatty liver disease (NAFLD) is known to adversely affect stroke recovery. However, few studies investigate how stroke elicits liver dysfunction, particularly, how stroke in type 2 diabetes mellitus (T2DM) exacerbates progression of NAFLD. In this study, we test whether exosomes harvested from human umbilical cord blood (HUCBC) derived CD133 + cells (CD133 + Exo) improves neuro-cognitive outcome as well as reduces liver dysfunction in T2DM female mice. Methods: Female, adult non-DM and T2DM mice subjected to stroke presence or absence were considered. T2DM-stroke mice were randomly assigned to receive PBS or Exosome treatment group. CD133 + Exo (20 µg/200 µl PBS, i.v.) was administered once at 3 days after stroke. Evaluation of neurological (mNSS, adhesive removal test) and cognitive function [novel object recognition (NOR) test, odor test] was performed. Mice were sacrificed at 28 days after stroke and brain, liver, and serum were harvested. Results: Stroke induces severe and significant short-term and long-term neurological and cognitive deficits which were worse in T2DM mice compared to non-DM mice. CD133 + Exo treatment of T2DM-stroke mice significantly improved neurological function and cognitive outcome indicated by improved discrimination index in the NOR and odor tests compared to control T2DM-stroke mice. CD133 + Exo treatment of T2DM stroke significantly increased vascular and white matter/axon remodeling in the ischemic brain compared to T2DM-stroke mice. However, there were no differences in the lesion volume between non-DM stroke, T2DM-stroke and CD133 + Exo treated T2DM-stroke mice. In T2DM mice, stroke induced earlier and higher TLR4, NLRP3, and cytokine expression (SAA, IL1ß, IL6, TNFα) in the liver compared to heart and kidney, as measured by Western blot. T2DM-stroke mice exhibited worse NAFLD progression with increased liver steatosis, hepatocellular ballooning, fibrosis, serum ALT activity, and higher NAFLD Activity Score compared to T2DM mice and non-DM-stroke mice, while CD133 + Exo treatment significantly attenuated the progression of NAFLD in T2DM stroke mice. Conclusion: Treatment of female T2DM-stroke mice with CD133 + Exo significantly reduces the progression of NAFLD/NASH and improves neurological and cognitive function compared to control T2DM-stroke mice.

Treatment With an Angiopoietin-1 Mimetic Peptide Improves Cognitive Outcome in Rats With Vascular Dementia.

Background and Purpose: Vascular dementia (VaD) is a complex neurodegenerative disease affecting cognition and memory. There is a lack of approved pharmacological treatments specifically for VaD. In this study, we investigate the therapeutic effects of AV-001, a Tie2 receptor agonist, in middle-aged rats subjected to a multiple microinfarct (MMI) model of VaD. Methods: Male, 10-12 month-old, Wistar rats were employed. The following experimental groups were used: Sham, MMI, MMI+1 µg/Kg AV-001, MMI+3 µg/Kg AV-001, MMI+6 µg/Kg AV-001. AV-001 treatment was initiated at 1 day after MMI and administered once daily via intraperitoneal injection. An investigator blinded to the experimental groups conducted a battery of neuro-cognitive tests including modified neurological severity score (mNSS) test, novel object recognition test, novel odor recognition test, three chamber social interaction test, and Morris water maze test. Rats were sacrificed at 6 weeks after MMI. Results: There was no mortality observed after 1, 3, or 6 µg/Kg AV-001 treatment in middle-aged rats subjected to MMI. AV-001 treatment (1, 3, or 6 µg/Kg) does not significantly alter blood pressure or heart rate at 6 weeks after MMI compared to baseline values or the MMI control group. Treatment of MMI with 1 or 3 µg/Kg AV-001 treatment does not significantly alter body weight compared to Sham or MMI control group. While 6 µg/Kg AV-001 treated group exhibit significantly lower body weight compared to Sham and MMI control group, the weight loss is evident starting at 1 day after MMI when treatment was initiated and is not significantly different compared to its baseline values at day 0 or day 1 after MMI. AV-001 treatment significantly decreases serum alanine aminotransferase, serum creatinine, and serum troponin I levels compared to the MMI control group; however, all values are within normal range. MMI induces mild neurological deficits in middle-aged rats indicated by low mNSS scores (<6 on a scale of 0-18). Compared to control MMI group, 1 µg/Kg AV-001 treatment group did not exhibit significantly different mNSS scores, while 3 and 6 µg/Kg AV-001 treatment induced significantly worse mNSS scores on days 21-42 and 14-42 after MMI, respectively. MMI in middle-aged rats induces significant cognitive impairment including short-term memory loss, long-term memory loss, reduced preference for social novelty and impaired spatial learning and memory compared to sham control rats. Rats treated with 1 µg/Kg AV-001 exhibit significantly improved short-term and long-term memory, increased preference for social novelty, and improved spatial learning and memory compared to MMI rats. Treatment with 3 µg/Kg AV-001 improves short-term memory and preference for social novelty but does not improve long-term memory or spatial learning and memory compared to MMI rats. Treatment with 6 µg/Kg AV-001 improves only long-term memory compared to MMI rats. Thus, 1 µg/Kg AV-001 treatment was selected as an optimal dose. Treatment of middle-aged rats subjected to MMI with 1 µg/Kg AV-001 significantly increases axon density, myelin density and myelin thickness in the corpus callosum, as well as increases synaptic protein expression, neuronal branching and dendritic spine density in the cortex, oligodendrocytes and oligodendrocyte progenitor cell number in the cortex and striatum and promotes neurogenesis in the subventricular zone compared to control MMI rats. Conclusions: In this study, we present AV-001 as a novel therapeutic agent to improve cognitive function and reduce white matter injury in middle aged-rats subjected to a MMI model of VaD. Treatment of MMI with 1 µg/Kg AV-001 significantly improves cognitive function, and increases axon density, remyelination and neuroplasticity in the brain of middle-aged rats.

TNFα Triggers an Augmented Inflammatory Response in Brain Neurons from Dahl Salt-Sensitive Rats Compared with Normal Sprague Dawley Rats.

Tumor Necrosis Factor (TNF)-α is a proinflammatory cytokine (PIC) and has been implicated in a variety of illness including cardiovascular disease. The current study investigated the inflammatory response trigged by TNFα in both cultured brain neurons and the hypothalamic paraventricular nucleus (PVN), a key cardiovascular relevant brain area, of the Sprague Dawley (SD) rats. Our results demonstrated that TNFα treatment induces a dose- and time-dependent increase in mRNA expression of PICs including Interleukin (IL)-1ß and Interleukin-6 (IL6); chemokines including C-C Motif Chemokine Ligand 5 (CCL5) and C-C Motif Chemokine Ligand 12 (CCL12), inducible nitric oxide synthase (iNOS), as well as transcription factor NF-kB in cultured brain neurons from neonatal SD rats. Consistent with this finding, immunostaining shows that TNFα treatment increases immunoreactivity of IL1ß, CCL5, iNOS and stimulates activation or expression of NF-kB, in both cultured brain neurons and the PVN of adult SD rats. We further compared mRNA expression of the aforementioned genes in basal level as well as in response to TNFα challenge between SD rats and Dahl Salt-sensitive (Dahl-S) rats, an animal model of salt-sensitive hypertension. Dahl-S brain neurons presented higher baseline levels as well as greater response to TNFα challenge in mRNA expression of CCL5, iNOS and IL1ß. Furthermore, central administration of TNFα caused significant higher response in CCL12 in the PVN of Dahl-S rats. The increased inflammatory response to TNFα in Dahl-S rats may be indicative of an underlying mechanism for enhanced pressor reactivity to salt intake in the Dahl-S rat model.

Activation of Orexin System Stimulates CaMKII Expression.

Hyperactivity of the orexin system within the paraventricular nucleus (PVN) has been shown to contribute to increased sympathetic nerve activity (SNA) and blood pressure (BP) in rodent animals. However, the underlying molecular mechanisms remain unclear. Here, we test the hypothesis that orexin system activation stimulates calcium/calmodulin-dependent kinase II (CaMKII) expression and activation, and stimulation of CaMKII expressing PVN neurons increases SNA and BP. Real-time PCR and/or western blot were carried out to test the effect of orexin-A administration on CaMKII expression in the PVN of normal Sprague Dawley (SD) rats and orexin receptor 1 (OX1R) expressing PC12 cells. Immunostaining was performed to assess OX1R cellular localization in the PVN of SD rats as well as orexin-A treatment on CaMKII activation in cultured hypothalamic neurons. In vivo sympathetic nerve recordings were employed to test the impact of optogenetic stimulation of CaMKII-expressing PVN neurons on the renal SNA (RSNA) and BP. The results showed that intracerebroventricular injection of orexin-A into the SD rat increases mRNA expression of CaMKII subunits in the PVN. In addition, Orexin-A treatment increases CaMKII expression and its phosphorylation in OX1R-expressing PC12 cells. Furthermore, Orexin-A treatment increases CaMKII activation in cultured hypothalamic neurons from neonatal SD rats. Finally, optogenetic excitation of PVN CaMKII-expressing neurons results in robust increases in RSNA and BP in SD rats. Our results suggest that increased orexin system activity activates CaMKII expression in cardiovascular relevant regions, and this may be relevant to the downstream cardiovascular effects of CaMKII.

Activation of Orexin 1 Receptors in the Paraventricular Nucleus Contributes to the Development of Deoxycorticosterone Acetate-Salt Hypertension Through Regulation of Vasopressin.

Salt-sensitivity is a major factor in the development of hypertension. The brain orexin system has been observed to play a role in numerous hypertensive animal models. However, orexin's role in the pathology of salt-sensitive hypertension (SSH) remains to be adequately explored. We assessed the impact of orexin hyperactivity in the pathogenesis of the deoxycorticosterone acetate (DOCA) - salt rat model, specifically through modulation of Arginine Vasopressin (AVP). Adult male rats were separated into three groups: vehicle control, DOCA-salt, and DOCA-salt+OX1R-shRNA. DOCA-salt rats received subcutaneous implantation of a 21-day release, 75 mg DOCA pellet in addition to saline drinking water (1% NaCl and 0.2% KCl). DOCA-salt+OX1R-shRNA rats received bilateral microinjection of AAV2-OX1R-shRNA into the paraventricular nucleus (PVN) to knockdown function of the Orexin 1-Receptor (OX1R) within that area. Following 2-week to allow full transgene expression, a DOCA pellet was administered in addition to saline drinking solution. Vehicle controls received sham DOCA implantation but were given normal water. During the 3-week DOCA-salt or sham treatment period, mean arterial pressure (MAP) and heart rate (HR) were monitored utilizing tail-cuff plethysmography. Following the 3-week period, rat brains were collected for either PCR mRNA analysis, as well as immunostaining. Plasma samples were collected and subjected to ELISA analysis. In line with our hypothesis, OX1R expression was elevated in the PVN of DOCA-salt treated rats when compared to controls. Furthermore, following chronic knockdown of OX1R, the hypertension development normally induced by DOCA-salt treatment was significantly diminished in the DOCA-salt+OX1R-shRNA group. A concurrent reduction in PVN OX1R and AVP mRNA was observed in concert with the reduced blood pressure following AAV2-OX1R-shRNA treatment. Similarly, plasma AVP concentrations appeared to be reduced in the DOCA-salt+OX1R-shRNA group when compared to DOCA-salt rats. These results indicate that orexin signaling, specifically through the OX1R in the PVN are critical for the onset and maintenance of hypertension in the DOCA-salt model. This relationship is mediated, at least in part, through orexin activation of AVP producing neurons, and the subsequent release of AVP into the periphery. Our results outline a promising mechanism underlying the development of SSH through interactions with the brain orexin system.

Guipi decoction for coronary heart disease: A protocol for a systematic review and meta-analysis.

BACKGROUND: Coronary heart disease (CHD) is the leading cause of morbidity and mortality worldwide. Guipi Decoction (GPD) is a classical traditional Chinese medication used to treat CHD. However, systematic review and meta-analysis regarding its efficacy and safety has not been systematically evaluated. The objective of this protocol is to determine the efficacy and safety of GPD in the treatment of CHD. METHODS: Randomized controlled trials evaluating the effectiveness and safety of GPD in the treatment of CHD will be retrieved from 8 electronic databases, including PubMed, EMBASE, Cochrane Library, Web of science, China National Knowledge Infrastructure Database, VIP Database, Wanfang Database and China Biology Medicine Database. Study selection, data collection, risk of bias assessment, and evaluation of the quality of evidence will be performed in order. Data will be analyzed by RevMan V.5.3.5 software. RESULTS: This study will evaluate the efficacy and safety of GPD in the treatment of CHD. CONCLUSION: This systematic review will provide evidence for determining whether or not GPD is an effective and safe intervention for CHD. PROSPERO REGISTRATION NUMBER: PROSPERO CRD 42020156420.

Fuling Sini decoction for patients with chronic heart failure: A protocol for a systematic review and meta-analysis.

BACKGROUND: Chronic heart failure (CHF) is one of the most important cardiovascular diseases worldly, with high morbidity and mortality. Fuling Sini decoction (FSD) has been used in the management of CHF widely in China, while its effective evidence is not clear. A systematic review and meta-analysis to evaluate FSD for CHF is lacking. Hence, we propose a protocol for systematic evaluation of its efficacy and safety for CHF. METHODS: We will search the following electronic databases from inception to October 2018, including EMBASE, Cochrane Center Registration Controlled trials (Cochrane Library), PubMed, Medline, WHO International Clinical Trials Registry Platform, China Biomedical Literature Database (CBM), China National Knowledge Infrastructure (CNKI), Chinese Scientific Journal Database (VIP), and Wan-fang database. Only randomized controlled trials (RCTs) of FSD alone or combined with other management for CHF will be included. Two independent reviewers will screen the literature and extract data according to the Cochrane Handbook method. The assessment of bias risk, data synthesis, subgroup analysis, and meta-analyses and finally meta-analysis will be performed by using RevMan V.5.3.5 software. RESULTS: This systematic review will provide high-quality evidence and may be the first to evaluate efficacy and safety of FSD in the treatment of CHF. CONCLUSION: This systematic review will provide evidence for judging whether FSD is an effective management for CHF or not. PROSPERO REGISTRATION NUMBER: CRD 42018110210.