bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of adenosine in muscle afferent TRPV1‐engaged reflex sympathetic response
Authors: Li Jianhua , Xing Jihong , Sinoway Lawrence
Published: June 2021

Capsaicin injected into the arterial blood supply of the hindlimb muscles of rats increases the renal sympathetic nerve activity (RSNA) and blood pressure via a reflex mechanism. The responses are altered in disease states such as heart failure and hindlimb muscle ischemia. The purpose of this study was to examine the role adenosine plays in modulating metabolite‐sensitive receptor potential vanilloid type 1 (TRPV1) receptors/capsaicin receptor activity. Our results show that the prior application of 1 μM of adenosine significantly attenuated capsaicin‐induced current of the dorsal root ganglion neurons innervating muscles (current amplitude: 2.37±0.35 nA in 19 control cells and 0.56±0.15 nA in 19 cells with prior adenosine, P<0.05). 100 µM adenosine injected into the femoral artery had an inhibitory effect on the reflex RSNA response induced by capsaicin in six rats. Arterial injection of adenosine analogue CGS21680 (A2A subtype receptor agonist, 100 µM) significantly attenuated the reflex response in six rats. Based on these data, we speculate that adenosine is likely to inhibit muscle afferent TRPV1‐engaged reflex responses as the levels of adenosine are increased in the hindlimb muscles and/or its receptors. The role this system plays in pathologic conditions needs to be studied. (All procedures outlined in this study were performed in compliance with the rules and regulations described in the NIH Guide for the Care and Use of Laboratory Animals).
Activation of NTS A1 adenosine receptors impairs cardiopulmonary chemoreflex responses of adrenal (ASNA), lumbar (LSNA) and renal (RSNA) sympathetic nerve activity
Authors: Ichinose Tomoko K , O'Leary Donal S , Scislo Tadeusz J
Published: June 2021

Activation of NTS A1 adenosine receptors evokes differential sympathoactivation (ASNA>RSNA≥LSNA). These responses were attenuated/abolished by sinoaortic denervation plus vagotomy or blockade of glutamatergic transmission in the NTS suggesting that these receptors may inhibit baroreflex transmission in the NTS (Am J Physiol 283: H1588‐99, 2002; 294: H172‐82, 2008). However, potential inhibition of cardiopulmonary chemoreceptors may also contribute to the A1 adenosine‐receptor‐mediated differential regional sympathoactivation. Therefore, in the present study we compared regional sympathoinhibition evoked by right atrial injections of 5HT3 receptor agonist (phenylbiguanide (PBG) 1‐8 μg/kg) before and after stimulation of NTS A1 adenosine receptors (microinjections of CPA, 330 pmol/50 nl) in urethane/chloralose anesthetized rats (n=7). Activation of cardiopulmonary chemoreceptors evoked differential, dose dependent sympathoinhibition (RSNA>ASNA>LSNA). The responses to all doses of PBG (Δ%) were greatly attenuated following stimulation of NTS A1 adenosine receptors (‐95.6±1.2 vs. ‐40.4±8.5, ‐75.4±5.8 vs. ‐27.5±6.8, ‐60.9±3.3 vs. ‐29.1±7.3, for RSNA, ASNA and LSNA respectively at the maximal dose of PBG). We conclude that activation of A1 adenosine receptors in the NTS differentially inhibits the cardiopulmonary chemoreflex responses of regional sympathetic outputs. NIH HL‐67814
Ischemic heart failure induces chronic sympathoexcitation in conscious Sprague Dawley rats.
Authors: Burris Sarah Kimberly , Knuepfer Mark M
Published: June 2021

Indirect estimates of sympathetic nerve activity (SNA) in rats suggest increased sympathetic drive contributes to the compensatory remodeling and progression of heart failure following myocardial infarction. We examined the role of SNA during the development of ischemic heart failure by recording SNA for weeks in conscious rats. Male Sprague‐Dawley rats were instrumented to record arterial pressure, heart rate, cardiac output, electrocardiographic activity, and adrenal and renal SNA. Cardiac function and size of the infarct were assessed pre‐ and post‐infarct using 2‐D and M‐mode echocardiography and/or histology. Ganglionic blockade (trimethaphan, 10 mg/kg, i.v.) was administered daily to ensure quality of SNA recordings. Responses to acute stress (cold pressor test) were recorded prior to and for 2‐3 weeks following coronary ligation. Greater increases in resting renal and adrenal SNA were seen following myocardial infarction. Acute stress with cold water resulted in consistent increases in SNA and pressor responses in which SNA responses were attenuated following myocardial infarction. Our findings directly demonstrate that resting SNA increases during the development of ischemic heart failure yet reflex responses are blunted. We suggest that chronic sympathoexcitation contributes to the progression of heart failure. Supported by USPHS DA0017371, HL091440 & USPHS T32 GM008306.
ANGIOTENSIN‐(1‐7) ICV CHRONIC INFUSION IMPROVES BAROREFLEX CONTROL OF RENAL SYMPATHETIC NERVE ACTIVITY IN DOCA‐SALT HYPERTENSIVE RATS
Authors: Guimarães Priscila S , Xavier Carlos H , Fontes Marco AP , Santos Robson AS , Campagnole‐Santos Maria J
Published: June 2021

We evaluated the effect of angiotensin‐(1‐7) [Ang‐(1‐7)] ICV chronic infusion in baseline and baroreflex control of renal sympathetic nerve activity (RSNA) in DOCA‐salt hypertensive rats. Sprague‐Dawley rats were submitted to uninephrectomy (UNX) or DOCA‐salt hypertension model (UNX + DOCA + saline). After 7 days, Ang‐(1‐7) ICV infusion (0.2 nmol/ 0.5 μl/ hour) or 0.9% sterile saline (0.5 μl/ hour) was initiated by osmotic mini‐pumps for 14 days. On the 21th day, the animals were anaesthetized (urethane; i.p.) and baseline arterial pressure (MAP), heart rate (HR) and RSNA, and baroreflex control RSNA were evaluated. As expected, DOCA‐salt treatment induced an increase in baseline MAP (122 ± 3 mmHg, n=4 vs 93 ± 2 mmHg, n=5, UNX), reduced baroreflex control of RSNA (1.33 ± 0.10 %ΔRSNA/ mmHg, n=4 vs 2.73 ± 0.29 %ΔRSNA/ mmHg, n=5, UNX), without any changes in baseline HR or RSNA (130 ± 17 spikes/sec, n=5 vs 118 ± 12 spikes/sec, n=4 UNX). Animals submitted to Ang‐(1‐7) ICV infusion presented a reduced MAP (conscious rats) and a greater baroreflex control of RSNA (2.24 ± 0.20 %ΔRSNA/ mmHg, n=4), without any significant in baseline RSNA (113 ± 10 spikes/sec, n=4). These results showed that central Ang‐(1‐7) modulates baroreflex control of RSNA in DOCA‐salt rats, and suggest that chronic increase in Ang‐(1‐7) levels in the brain may attenuate high blood pressure, in part by improving baroreflex control of sympathetic activity.
TRPV1‐expressing Afferents Innervate the Aorta and Contribute to Baroreflex Control of Cardiovascular Function
Authors: Li De‐Pei , Sun Hao , Chen Shao‐Rui , Pan Hui‐Lin
Published: June 2021

The arterial baroreflex is important in the regulation of blood pressure and sympathetic outflow. The transient receptor potential vanilloid receptor 1 (TRPV1) is expressed on primary sensory neurons including baroreceptive neurons. Here, we tested the hypothesis that TRPV1‐expressing afferents participate in the control of sympathetic outflow. We used resiniferatoxin (RTX), an ultrapotent analogue of capsaicin, to remove TRPV1‐expressing neurons and afferent fibers in rats. Enhanced immunofluorescent staining showed that TRPV1‐immunoreactivities were present on the nerve endings in the ascending aorta and aortic arch, nodose ganglion (NG) neurons, and afferent nerve endings and neurons in the nucleus solitary tract (NTS). RTX treatment removed TRPV1 immunoreactivities in the aorta, NG neurons, and NTS. Renal sympathetic nerve activity (RSNA), arterial blood pressure (ABP), and heart rate (HR) were recorded in anesthetized rats receiving RTX treatment. The baroreflex was triggered by lowering and raising blood pressure via intravenous infusion of nitroglycerin and phenylephrine, respectively. The baroreflex control of RSNA and HR were impaired in high blood pressure range in RTX‐treated rats. Furthermore, the maximum gain of the baroreflex control of RSNA and HR were significantly decreased in RTX‐treated rats. Furthermore, blocking TRPV1 receptors with Iodo‐RTX blunted the baroreflex control of RSNA and HR and decreased the maximum gain of baroreflex function curves in high blood pressure range. These data provide important new evidence that TRPV1 receptors are expressed in afferent nerves and neurons along the baroreflex pathway. TRPV1‐expressing neurons contributes to the baroreflex control of the cardiovascular function.
Effect of Castration on Mesenteric Arterial and Portal Venous Responses to Splanchnic Nerve Stimulation
Authors: Martin Doug Stuart , Kjellsen Barton
Published: June 2021

Androgens exacerbate hypertension development. Our previous work suggested that androgens also amplify adrenergic constriction (Journal of Hypertension 23:2229). This study tested the hypothesis that male sex steroids modulate neural control of splanchnic resistance and capacitance function.

Five week old male spontaneously hypertensive rats (SHR) underwent sham operation or castration and were studied at 12‐16 weeks of age. Mean arterial blood pressure (MAP) and heart rate (HR) were recorded in conscious rats. The rats were then anesthetized and instrumented for splanchnic nerve stimulation and mesenteric arterial and portal venous blood flow recording. Frequency response curves (1‐12 Hz) were constructed before and after inhibition of N type calcium channels with omega conotoxin.

MAP was elevated in sham operated (151±6 mm Hg) compared to castrated SHR (135±3 mm Hg). Splanchnic nerve stimulation caused frequency dependent changes in mesenteric arterial and portal venous blood flow in both groups. However, the responses were greater in sham operated SHR compared castrated SHR. Treatment with omega conotoxin markedly attenuated the differences between groups.

Collectively, these data show that castration modulates neurally mediated splanchnic resistance and capacitance vascular responses. This effect may be mediated in part via the N type calcium channel. Supported by NIH #63053 and AHA #0515443Z.
Contributions of cardiac output and total peripheral resistance to vasovagal syncope during orthostasis in humans
Authors: Fu Qi , VanGundy Tiffany B , Shibata Shigeki , Galbreath M Melyn , Levine Benjamin D
Published: June 2021

Mean arterial pressure is determined by cardiac output (CO) and total peripheral resistance (TPR). However, the contribution of CO and TPR to blood pressure (BP) drop during vasovagal syncope in humans is uncertain. We studied retrospectively 26 subjects (4 men and 22 women, age range 16‐50) who had no previous history of syncope but had presyncopal episodes during 45 min 60° upright tilt in our lab in the past 4 years. Muscle sympathetic nerve activity (MSNA), heart rate (HR), and BP were recorded continuously, while beat‐to‐beat hemodynamics were derived from the Modelflow method calibrated by the acetylene rebreathing technique. Systolic BP decreased progressively and then rapidly 100 and 20 s prior to presyncope, while diastolic BP started to decrease 40 s before presyncope. Fifteen of them (58%, Group A) had falls in both CO and TPR, while 11 (42%, Group B) had only a fall in CO 60 s prior to presyncope. Cardiac index, calculated as CO divided by body surface area, was lower [2.10±0.39 (SD) L/min/m2 vs 2.50±0.53, P=0.0498], while TPR was greater (1.30±0.83 MU vs 0.87±0.16, P=0.0572) in Group B than Group A at presyncope. The steeper fall in CO in Group B was due to a decrease in HR rather than stroke volume. MSNA decreased rapidly 20 s prior to presyncope in both groups. These results suggest that both CO and TPR are determinant factors for the drop in arterial pressure. A marked decrease in CO alone or a modest decrease in CO accompanied by a modest systemic vasodilation contributes to syncope during orthostasis in humans.
HPLC is more sensitive to assess urinary albumin than nephelometry in acute stroke patients
Authors: Toth Peter , Marko Lajos , Halmai Richard , Bosnyak Edit , Szapary Laszlo , Koller Akos , Komoly Samuel , Nagy Judit , Wittmann Istvan
Published: June 2021

Microalbuminuria, the appearance of albumin in the urine, is one the first signs of a disease process in many pathological conditions. However, the definition or identification of "microalbuminuria" may depend on the sensitivity of methods used to measure urinary albumin. In the past, immuno‐nephelometry (NM) was used to routinely detect immunoreactive albumin. However, high‐performance liquid chromatography (HPLC) detects both immunoreactive and non‐immunoreactive albumins. Early recognition of microalbuminuria is important, because recent studies have shown that it is associated with elevated risk of stroke. We hypothesized, that use of HPLC will detect microalbuminuria in more patients with acute stroke than NM. Thus in ischemic stroke patients (n=100) the albumin to creatinine ratio was measured by HPLC and NM to characterize microalbuminuria. We have found that HPLC detected albumin in the urine of 81,8% of stroke patients, whereas NM detected it only in 46,5%. The concentration of urinary albumin detected by HPLC was significantly greater than by nephelometry (HPLC: 203,8±37 NM: 106±27 mg/mmol; p<0,001). In conclusion, HPLC can detect microalbuminuria in more stroke patients and perhaps earlier than NM can. Thus use of HPLC seems to be preferable for screening stroke patient for microalbuminuria.

Grants: Hungarian Sci. Res. Funds/OTKA T48376, K71591, F68638; Health Sci. Council/ETT 364/2006, Hungarian Neuroimaging Foundation, AHA F. Aff. 0855910D, USA
The Effects of High Glucose Concentrations on Cerebral Myogenic Tone of Control and Type II Diabetic Rats
Authors: Kelly Aisha Imani , Elgebaly Mostafa M. , Ergul Adviye
Published: June 2021

Changes in cerebrovascular myogenic reactivity can hamper autoregulatory mechanisms responsible for maintaining blood flow to the brain. Previous studies report that in vitro exposure to high glucose reduces the myogenic tone of isolated cerebral arteries from non‐diabetic animals and causes vasodilatation. However, the effect of buffer glucose concentration on myogenic reactivity of isolated cerebral vessels from diabetic animals with preexisting vascular disease is unknown. We hypothesized that myogenic responses of the middle cerebral artery (MCA) to increasing perfusion pressure in normal and high glucose containing buffer differ in control and diabetic animals. MCAs from aged‐matched Wistar (n= 10) and type II diabetic Goto‐Kakizaki (GK) rats (n=3,4) were placed in buffer containing either 5mM or 25mM glucose and pressurized using an arteriograph system. All groups maintained their myogenic properties across the autoregulatory range (40‐120 mmHg) and constricted in response to increasing pressure. At 80 mmHg, diabetic MCAs in 5mM glucose had higher tone than control group (29.3± 5.7% vs 17.2±3.5%). GK rats tended to have lower myogenic tone in 25mM glucose whereas control animals showed increased tone as compared to 5mM glucose conditions across the pressure range. There was no difference in lumen diameter or media thickness among the groups. Therefore, in vitro vascular reactivity is affected by buffer glucose concentration; adjusting it to reflect animal blood glucose levels may allow better evaluation of myogenic reactivity. NIH DK074385, DK074385‐04S1 and AHA 0740002NM
Vascular Response in Mice Deficient in the Potassium Channel, TREK‐1
Authors: Namiranian Khodadad , Lloyd Eric E , Marrelli Sean P , Bryan Robert M
Published: June 2021

TREK‐1, a member of the two‐pore domain potassium channel family, is highly expressed in the vascular system. We hypothesize that TREK‐1 is involved with regulating the diameter of cerebral vessels. A TREK‐1 knockout mouse (KO) was generated by replacing the first two coding exons of TREK‐1 with a neomycin/beta‐galactosidase cassette. KO mice had normal blood pressure and cardiac function. Vascular responses were measured in isolated basilar arteries (BA) and middle cerebral arteries (MCA). Linoleic acid (LA, 10 μM and 100 μM), an activator of TREK‐1 and Ca‐activated K channels (BKCa), dilated pre‐constricted BA of wild‐type (WT) and KO mice to a similar extent. However, after selectively blocking BKCa with penitrem A, the dilation to LA in BA from KO mice was reduced 19±5% (n=6), while it was not affected in WT. This suggests that part of LA‐induced dilation is mediated via TREK‐1. Acetylcholine‐mediated dilations in BA and ATP‐mediated dilations in MCA of KO mice were not different from WT mice, either before or after inhibition of nitric oxide synthase. We conclude that TREK‐1 can regulate diameter of cerebral arteries; however, it does not appear to be involved with certain endothelial mediated dilations. This project is supported by a Predoctoral Fellowship 0815342F (KN) and grant 0665100Y (SPM) from American Heart Association, and NIH grants RO1 NS‐46666 (RMB), PO1 NS‐038660 (RMB), RO1 HL‐088435 (SPM).
Role of cAMP and cGMP in arachidonic acid and PGE2 ‐induced cerebral vasodilation in piglets
Authors: Kanu Alie , Leffler Charles W
Published: June 2021

Arachidonic acid (AA) products are important contributors to regulation of newborn cerebral circulation. PGE2 stimulates cAMP production in cultured piglet cerebrovascular smooth muscle cells. PGE2 increases cAMP and cGMP in CSF. Therefore, it is possible that AA and its metabolite, PGE2, generated in cerebral vessels produce vasodilation by increasing cGMP and cAMP production. The aim of this study was to determine if cAMP and/or cGMP mediate the vasodilatory effects of AA and PGE2. Experiments were performed in anesthetized piglets with closed cranial windows. In experiments with PGE2, piglets were administered indomethacin (5mg/kg, iv) to inhibit endogenous prostanoid production. Pial arteriolar responses to topical application of AA (10−7 M, 10−5 M), PGE2 (10−7 M, 10−5 M) or vehicle were determined without and with the guanylyl cyclase inhibitor ODQ (10−5 M) or the adenylyl cyclase inhibitor SQ22536 (10−6 M). ODQ (2.5x10−5 M) blocked AA‐ and PGE2‐induced cerebral vasodilation. SQ22536, that blocked the dilation to the adenylyl cyclase activator, Forskolin (10−7 M), also inhibited AA‐ and PGE2‐induced cerebral vasodilation. These data suggest that both cGMP and cAMP mediate vasodilator effects of AA and PGE2. Since both ODQ and SQ22536 blocked the cerebral vasodilator effect of AA and PGE2, cAMP and cGMP may act in a sequential manner. Studies to address this subject are in progress.
Hyperglycemia increases myogenic tone and the dilatory effects of peroxynitrite (ONOO‐) in cerebral arteries: Role of nitrotyrosine (NT) and reactive oxygen species (ROS)
Authors: Cipolla Marilyn J. , Godfrey Julie A. , Smith Jeremiah
Published: June 2021

We investigated the vasoactive effects of ONOO‐ in cerebral arteries and tested the hypothesis that enhanced ROS during hyperglycemia causes greater vasodilation due to increased NT accumulation in vascular smooth muscle (VSM). Posterior cerebral arteries (PCA) were isolated from male Wistar rats that were normoglycemic (NG, n=8), hyperglycemic for 3 days by STZ injection (50 mg/kg; HG, n=6), or HG and treated with the ROS scavenger Tempol (100 mg/kg; HG+Temp, n=8). NT in VSM was determined by confocal microscopy. Blood glucose was increased in HG and HG+Temp animals vs. NG (mg/dL): 334 ± 11 and 312 ± 22 vs. 137 ± 3 (p<0.01). PCA from HG rats had increased myogenic tone vs. NG at 75 mmHg (42 ± 3 vs. 27 ± 4%; p<0.01) that was not ameliorated by Tempol (45 ± 3%). PCA from NG and HG animals dose‐dependently constricted to ONOO‐ from 0.01 to 1.0 μM; however, at 3.0 μM ONOO‐, PCA from HG animals dilated 11% whereas PCA from NG animals constricted 3% further. Treatment of HG animals with Tempol prevented the dilation to ONOO‐. NT content in VSM was increased in PCA from HG vs. NG animals, but was restored to NG levels by Tempol. NT content in VSM for NG, HG and HG+Temp was (intensity/μm2) 8.4 ± 5.1, 37.0 ± 4.7 and 16.5 ± 5.6 (p<0.05 vs. HG). These results suggest that oxidative stress associated with a hyperglycemic environment promotes vasodilation at lower concentrations of ONOO‐, possibly due to NT accumulation in VSM. Supported by NINDS NS043316.
Decreased focal cerebral ischemic injury in Tek‐Grin1 KO mice
Authors: Alexander Jonathan Steven , Yilmaz Gokhan , Granger D Neil , Minagar Alireza , Jennings Merilyn H
Published: June 2021

Prior reports by our and other labs suggests that NMDA receptors in vascular endothelial cells mediate stroke‐like forms of injury in vitro, but equivalent demonstrations of vascular NMDA receptor participation in stroke in vivo are controversial. To overcome these problems, NMDAR1 (Grin1)/Tek (Tie‐2)‐ Cre mice, a mouse endothelial‐selective knockout for NMDAR1 were used in a model of stroke, middle cerebral artery occlusion (MCAO). Tek‐Grin‐/‐ mice showed significantly less neurological injury (p<0.05)and infarct size (p<0.05) measured at 24h (after 1h of middle cerebral artery occlusion). These results provide additional evidence in support of the role of NMDARs in post‐ischemic vascular stress in stroke.
Astrocyte‐derived glutathione attenuates hemin‐induced cytotoxicity in murine cerebral microvessel
Authors: Laird Melissa Dee , Ramesh Sangeetha Sukumari , Alleyne Cargill H , Dhandapani Krishnan M
Published: June 2021

Intracerebral hemorrhage (ICH) induces neurovascular injury following the rupture of a blood vessel within the brain via poorly defined mechanisms. The aim of the present study was to determine whether glio‐vascular communication may restrict hemorrhagic vascular injury. Hemin concentration‐ and time‐dependently increased oxidative cytotoxicity in mouse bEND.3 microvessel cells. Cell death was preceded by a NFκB‐mediated increase in inflammatory gene expression, including up‐regulation of inducible nitric oxide synthase (iNOS) expression and activity. The inhibition of iNOS or the addition of a peroxynitrite decomposition catalyst reduced cell death. Interestingly, co‐treatment with astrocyte conditioned media (ACM) reversed hemin‐induced NF ?ÛB activation, nitrotyrosine formation, and apoptotic cell death via the release of the endogenous antioxidant, reduced glutathione (GSH). Prior treatment of astrocytes with the glutathione‐depleting agent, DL‐buthionine (S,R)‐sulfoximine (BSO) or direct addition of diethyl maleate, a thiol depleting agent, to ACM reversed the observed protection. In contrast, the addition of exogenous GSH or the GSH precursor, N‐acetylcysteine, were protective in bEND.3 cells. These data identify a previously unrecognized vasculoprotective role for astrocyte‐derived GSH and indicate therapeutic targeting of GSH may reduce neurovascular injury following ICH.
Janus Kinase 2 and Tissue Inhibitor of Matrix Metalloproteinase‐1 Mediate the Protective Effects of Erythropoietin in In‐Vitro Model of Hypoxia Ischemia
Authors: Souvenir Rhonda , Fathali Nancy , Tong Wenni , Lekic Tim , Chee Priscilla , Zhang John
Published: June 2021

Studies have shown that erythropoietin (EPO) therapy significantly reduced brain injury and improved neurological function in rat model of neonatal hypoxia ischemia. Studies have shown that increase matrix metalloproteinase‐9 (MMP‐9) contributes to hypoxia ischemic brain injury. This study investigated if tissue inhibitor of matrix metalloproteinase‐1 (TIMP‐1) and its upstream signaling molecule Janus kinase‐2 (JAK‐2) are involved in EPO‐induced neuroprotection in an in vitro model of hypoxia ischemia.

Hypoxia ischemia was induced in neuronal growth factor differentiated PC12 cells by 2 hours of oxygen and glucose deprivation. The cells were treated immediately before and during hypoxia ischemia with either: EPO, EPO + AG490 (JAK‐2 inhibitor), AG490, EPO + TIMP‐1 antibody, or TIMP‐1 antibody. Cell death, phosphorylation of JAK‐2, signal transducers and activators of transcription protein‐3 (STAT‐3), TIMP‐1 expression, and MMP‐9 activity were measured and compared with normoxic group.

Our data showed that EPO significantly increased cell survival, associated with increased TIMP‐1 expression, phosphorylation of JAK‐2, STAT‐3, and decreased MMP‐9 activity. These protective effects of EPO were significantly reduced by inhibition of JAK‐2 and TIMP‐1. Conclusion: JAK‐2 and TIMP‐1 are necessary for EPO‐induced neuroprotection hypoxia ischemia injury. Supported by NIH, grant NS052492.
Activation of GABA¬A receptor Protects Mitochondria and Reduces Cerebral ischemia.
Authors: Tyagi Neetu , Givvimani Srikanth , Kumar Munish , Kundu Soumi , Gillespie William M , Mishra Paras , Sathnur Pushpa Kumar , Lominadze David , Sen Utpal , Tyagi Suresh C
Published: June 2021

Gamma‐aminobutyric acid is the inhibitory neurotransmitter and has been identified in mitochondria. Because the release of mitochondrial factors is known to correlate with acute neurological damage, we proposed to investigate the possible neuroprotective effects of GABA‐A receptors in cerebral ischemia. In this study transient ischemia was induced in mice by occlusion in the middle of the carotid artery (MACO). Effects of GABA‐A receptor agonist (muscimol) were first studied in oxygen/glucose deprivation‐induced death of neuronal cells. Mitochondrial membrane potential, cytochrome‐c release, caspase 3 activation, the infarct volume and neurological function were examined in the absence or presence of muscimol. Our results shows that GABA‐A receptor agonist inhibits oxygen/glucose deprivation‐induced cell death, loss of mitochondrial membrane potential, downstream release of cytochrome‐C, which activates the caspase apoptotic signaling cascade. Furthermore, it decreases infarct size and improves neurological scores after MACO in mice. This study indicated that GABA‐A receptor is neuroprotective against cerebral ischemia. It also suggested mitochondrial permeability transition might be a valuable therapeutic target for acute neurodegeneration.

Supported by NIH grant # HL‐71010 & NS‐51568 to SCT.
Combinatorial treatment with progesterone and thyrotropin releasing hormone improves outcomes after traumatic brain injury
Authors: VanLandingham Jacob Wade , Rich Nicholas , Winnard Kenneth , Levenson Cathy W. , Figueiroa Sylvia , Schepkin Victor D. , Seth Rohit , Overton James M. , Stein Donald G.
Published: June 2021

Previous work has shown that the neurosteroid progesterone (PROG) and hypothalamic hormone thyrotropin releasing hormone (TRH) improve molecular and functional outcomes following traumatic brain injury (TBI) when used independently. This work was designed to evaluate if a combination of PROG and TRH results in further improvements in outcomes. Adult male Sprague‐Dawley rats were given a medial frontal cortex contusion using a weight‐drop device after craniotomy. After TBI, rats were treated with PROG (5 d), TRH (1 injection at 1 h post‐injury), or the combination of the two drugs (PROG+TRH). Uninjured sham‐operated and vehicle‐treated TBI rats served as controls. PROG+TRH treatment resulted in significant improvements in both sensory skills and learning and memory as measured by the tactile adhesive test and Morris Water Maze, respectively (n=11/group). Analysis of putative mechanisms responsible for these behavioral observations suggest that the tumor suppressor protein p53, the inflammatory marker TNF‐α, and GFAP‐positive reactive gliosis may all participate in the efficacy of this combinatorial treatment. Furthermore, preliminary use of diffusion magnetic resonance imaging at 21 Tesla (21T) suggests that diffusion MRI is a valuable novel tool in future determination of mechanisms and the ways to reduce water accumulation and edema after treatment.
Flurbiprofen Inhibits Glioblastoma Proliferation and Migration by a COX‐Independent Mechanism
Authors: Kohler Missia Elizabeth , Qadri Yawar , Fuller Catherine , Benos Dale
Published: June 2021

Glioblastomas (GBMs) are primary brain tumors that are difficult to treat. Non Steroidal Anti‐Inflammatory Drugs (NSAIDs) are a promising therapy for colon and prostate cancer. NSAIDs are described as non‐selective inhibitors of cyclooxygenase (COX) enzymes. While there is evidence that upregulation of COX‐2 in GBMs coincides with a worse prognosis for patients, it is unclear whether NSAIDs affect GBMs migration or proliferation through a COX‐mediated mechanism. This study examined whether a NSAID, flurbiprofen, affects GBMs through a COX‐dependent mechanism. COX‐1 and COX‐2 mRNA was detected in all GBM cell lines. Flurbiprofen decreased the proliferation of a GBM cell line in a dose dependent manner. This effect was seen at similar doses with both COX‐active and COX‐inactive enantiomers. GBM cell migration was measured using an electric cell‐substrate impedance sensing system, and migration was strongly inhibited by flurbiprofen in a non‐enantioselective manner. This effect on migration was recapitulated in transwell assays. Tests of the related drugs ibuprofen, carprofen, and ketoprofen indicate that carprofen has the greatest effect upon the migration of glioma cells. Ultimately, although COX‐1 and COX‐2 is present in GBMs, they do not appear to be involved in the effects of flurbiprofen on GBM migration and proliferation. This study was supported by NIH grant CA101952, the AMA Foundation, and the APS.
Identification of a novel neuronophagic cell in the partially denervated rat neurohypophysis
Authors: Lo David , Watt John
Published: June 2021

The objective of this study was to identify neuronophagic cells in the partially denervated neurohypophysis. This was accomplished by using a phagocytosis‐dependent labeling technique to visualize the incorporation of labeled axonal debris within neuronophagic cells in the neurohypophysis following unilateral transection of the hypothalamo‐neurohypophysial tract (HNT). Stereotaxic application of the biotin and rhodamine conjugated dextran amine Mini Ruby into the magnocellular neurons of the supraoptic nucleus led to conspicuous and stable labeling of the HNT. Following unilateral lesion of the HNT, we observed small rhodamine labeled granules within s100beta immunoreactive pituicytes, and also within immunophenotypically identified perivascular cells expressing the p75 low affinity neurotrophin receptor, the C3bi compliment receptor, and ciliary neurotrophic factor. At the ultrastructural level, we observed homogeneous labeling of magnocellular neurons and neurosecretory axons with Mini Ruby. In the partially denervated neurohypophysis, labeled axons were found to be enveloped by pituicyte processes as well as within the pituicyte cytoplasm. These results indicate that the anterograde degeneration induced by partial denervation of the rat neurohypophysis stimulates neuronophagia by both perivascular cells and pituicytes. This study was supported by COBRE 2P20RR017699‐06

Grant Funding Source

COBRE 2P20RR017699‐06
Vitamin E suppresses bactericidal activity of polymorphonuclear leukocyte enhanced by hypoxia
Authors: Wang Jong‐Shyan
Published: June 2021

This investigation elucidates how various hypoxic interventions influence bactericidal activity of polymorphonuclear leukocyte (PMN) following treatment with an antioxidant vitamin E. Forty sedentary males were randomly assigned to vitamin E (n=20) and placebo (n=20) groups. Our results demonstrated that exposure to 12% O2 in the placebo group increased urinary 8‐isoprostane level and decreased plasma total antioxidant content. Moreover, this hypoxic exposure increased chemotactic, phagocytic, and oxidative burst activities of PMN, accompanied by increased expressions of L‐selectin, LFA‐1, Mac‐1, FcγIIIBR, C1qRp and C5aR on PMN. However, exposure to 12% O2 in the vitamin E group did not influence the opsonic/complement receptor expressions and bactericidal cascades of PMN. Additionally, the circulatory redox status, PMN‐mediated bactericidal activity and the opsonic/complement receptors on PMN were constant following exposure to 15%, 18% or 21% O2 in the two groups. We conclude that exposure to 12% O2 promotes PMN bactericidal activity, possibly by elevating lipid peroxidation. However, this hypoxic effect on PMN bactericidal activity is ameliorated by pretreatment with vitamin E.
Mitochondrial content and function decrease in mouse diaphragm after chronic hypoxia
Authors: Gamboa Jorge Luis , Andrade Francisco H
Published: June 2021

Loss of aerobic capacity induced by chronic hypoxia (i.e. diminished mitochondrial content) has been partially attributed to reduced physical activity. That is true for the limb muscles; however, hypoxia increases the workload on the diaphragm and other respiratory muscles. Thus, we hypothesized that mitochondrial content and function would not change in the mouse diaphragm following chronic hypoxia. Male C57BL/6J mice were kept in normoxia (FiO2=21%, control) or normobaric hypoxia (FiO2=10%, hypoxia) for 4 weeks. Then, mice were killed and the diaphragm and triceps surae were collected for analysis. In the diaphragm the content of electron transport chain (ETC) proteins was significantly reduced after 4 weeks of hypoxia (decreases of 25 to 30% compared to control). Mitochondrial volume density decreased after 4 weeks of hypoxia (control 33.6±5.5% vs. hypoxia 26.8±6.7%, p=0.013). A significant reduction of the ADP‐stimulated mitochondrial respiration (state 3) was also observed in the hypoxia group (159.0±28.8 vs. 112.2±38.9 nmolO2/min/mg protein, p=0.017). By contrast, no change was found in ETC protein content in triceps surae. However, chronic hypoxia decreased respiration states 2 (control 41.2±4.1 vs. hypoxia 33.2±6.7 nmolO2/min/mg protein, p=0.033) and 3 (control 196.6±32.6 vs. hypoxia 135±43.1 nmolO2/min/mg protein, p=0.018). These results disprove our initial hypothesis. Despite the increased work on ventilation, chronic hypoxia is more detrimental to mitochondrial content and function in the diaphragm than in limb muscles.

Supported by NIH/NIGMS F31 GM084665
Hypoxic Exercise Training Enhances Vascular Hemodynamics and Oxygen Utilization in Skeletal Muscles
Authors: Wu Min‐Huan , Wang Jong‐Shyan
Published: June 2021

This study investigates how hypoxic exercise training impacts on vascular hemodynamic and O2 utilization in exercising skeletal muscles. Twelve sedentary males were trained on a bicycle ergometer at about 60%VO2max under 15% O2 air condition for 20 minutes per day, 5 days per week for 4 weeks. Vascular hemodynamics and O2 consumption and re‐oxygenation rate in gastrocnemius were determined by near infrared spectroscopy. Our results showed that 4 weeks of hypoxic exercise training improved subjects' aerobic fitness at ventilation threshold and maximal performance. Acute exposure to 12%O2 decreased arterial hyperemia and venous compliance, as well as, oxygen consumption and re‐oxygenation rate in gastrocnemius at pre‐training state. However, the suppressive effects in vascular hemodynamics and tissue O2 utilization in gastrocnemius by acute hypoxic exposure were ameliorated following the hypoxic exercise training. Therefore, we conclude that long‐term hypoxic exercise training can improve cardiopulmonary fitness and increase the tolerance to hypoxia‐limited O2 transport and utilization in skeletal muscles.
Increased mitochondrial capacity due to chronic oxidative stress is reduced with age
Authors: Marcinek David J , Smith Steven R. , Van Remmen Holly
Published: June 2021

Oxidative stress has been proposed to modulate mitochondrial function by reducing the coupling of oxidative phosphorylation (P/O) and inducing mitochondrial biogenesis. Here we test whether the effects of a chronic oxidative stress are age‐dependent. We compare in vivo mitochondrial metabolism and mitochondrial protein expression in wild‐type and Cu, Zn‐superoxide dismutase knockout mice (SOD1) at multiple ages. We used 31P NMR and optical spectroscopies to measure ATP and O2 fluxes in vivo. The 7‐month old SOD1 mice exhibited a significantly reduced mitochondrial P/O (P=0.05) and an increase in both maximal mitochondrial ATP production (ATPmax) (P=0.02) and O2 consumption (VO2max) (P=0.01). The greater in vivo mitochondrial capacity in these mice was accompanied by increased expression of electron transport chain proteins. In contrast, in the 20‐month old mice there was no difference in P/O, ATPmax, and VO2max between the wild‐type and SOD1 genotypes. The effect of the SOD1 knockout on electron transport chain protein expression was also reduced in the 20‐month old mice. These data suggest that in young adults the chronic oxidative stress results in changes in mitochondrial metabolism that lead to increased mitochondrial capacity, whereas this response is reduced by 20 months. Supported by NIH grants AG028455, AG022385, and the Ellison Medical Foundation.
Age‐related changes in fluorescent imaging of superoxide in skeletal muscle arterioles
Authors: Kluess Heidi A , Stone Audrey J , Rogers Steven C , Kavdia Mahendra
Published: June 2021

Previous studies suggest that oxidative stress plays an important role in vascular aging. One of the mechanisms by which this may occur is via the second messenger pathway for angiotensin II. However, this has not been previously investigated in 1A arterioles. The purpose of the study was to measure the angiotensin II induced superoxide production in skeletal muscle arterioles and to determine the age‐related response. Gastrocnemius 1A arterioles from F344 rats (2 months and 24 months old) were dissected out, cannulated, warmed to 37°C and pressurized to 90cmH2O in a tissue bath. Dihydroethidium (DHE) was used to detect superoxide. Following equilibration procedures, the arterioles were loaded with 5 μM DHE for 20 minutes. Images were recorded every 15 seconds for 15 minutes. Resting fluorescence was recorded and then angiotensin II (10−7 M) was added. Additionally, we performed a dose response curve for angiotensin II and DHE fluorescence. Treatment of arterioles with angiotensin II caused a significant increase in superoxide production and resulted in vasoconstriction of arteriole. These results suggest that angiotensin II can cause significant oxidative stress in vascular tissue.
Mitochondrial Ca2+ and Apoptosis in Multiple Myeloma
Authors: Lynch Ronald , McGrath Ryan , Larson Karl , Escalante Aluvia , Jefferson Andrew , Landowski Terry
Published: June 2021

Multiple myeloma is a hematological cancer of plasma cells. Proteasome inhibitors, such as Bortezomib (BZ, Velcade/PS‐341) are used to treat myeloma because they induce apoptosis via activation of ER stress. We showed that ER stress results in a transient increase in cytosolic calcium ([Ca2+]c), and subsequent initiation of apoptosis. Since inhibition of mitochondrial Ca2+ ([Ca2+]m) uptake with ruthenium red blocked apoptotic initiation and activation of caspase activity, we analyzed [Ca2+]m in the myeloma line 8226 using Rhod‐2. Although BZ elicits a transient increase in [Ca2+]c, [Ca2+]m was found to rapidy (< 1 min) decrease. Following this initial decrease, two distinct responses were identified; either [Ca2+]m returned to then exceeded baseline, or [Ca2+]m remained below baseline levels. Our results indicate that there are two discrete sub‐populations of myeloma cells with respect to response to BZ which is consistent with the observed efficacy of BZ on <70% of the myeloma population. We propose that the population in which [Ca2+]m remains low is refractory to BZ induced apoptosis. Current studies will test this hypothesis, and aim to further characterize these two sub‐populations of myeloma cells. These data will allow us to identify targets for initiating apoptosis in the BZ refractory population with the goal of increasing the efficacy and specificity of drug treatment.
Pulmonary intravascular coagulation in aggregate anaphylaxis
Authors: Ryan James W.
Published: June 2021

Intravascular coagulation occurs within min. of i.v. ovalbumin (OA) challenge of an OA ‐sensitized guinea pig (GP). Microvascular obstruction of lungs is a leading cause of early death. Multiple indicator dilution studies indicate that up to 95% of pulmonary microvessels are occluded within 10 min. To help quantify intravascular coagulation, we examined the fate of 125I‐fibrinogen (I‐FN) within the first 10 min. of antigen challenge. Nine sensitized (ST) and four non‐sensitized (SC) GPs were given i.v. I‐FN, 3.5‐10 microCi, 10 min. before or with OA, 2 mg. Lungs were collected 10 min later. Hematocits of SC were unchanged pre‐ and post‐challenge, 0.456 v 0.464, but increased in ST, 0.442 v 0.629, p<0.0001. Lung wgt as % of body wgt increased in ST v SC: 0.871 v 0.542, p<0.0001. Coagulable 125I in plasma collected before and after challenge was unchanged in SC and ST: 90.3 v 91.0, and 90.2 v 90.2%. Trapped plasma in ST lungs was equivalent to 0.61 ml v 0.21 ml in SC lungs; p<0.01. Plasma vol. pre‐challenge did not differ between ST and SC groups; 32+ 0.05 ml/kg body weight. Taking intrinsic plasma fibrinogen concentration as 10 micromolar and fibrinogen MW as 340,000, total fibrin(ogen) in ST lungs 10 min after challenge was ~2.08 mg. Less than 5% was solubilized by homogenization of lungs. Our results suggest that widely diffuse fibrin plugs account for much of the pulmonary microvascular obstruction seen in aggregate anaphylaxis.
Optical stimulation of Channel Rhodopsin2(ChR2)‐transfected retrotrapezoid nucleus (RTN) neurons activates breathing
Authors: Abbott Stephen Bruce Gidley , Stornetta Ruth L , West Gavin H , Guyenet Patrice G
Published: June 2021

The role of the RTN neurons in breathing is not definitely established. RTN neurons are Phox2b‐expressing, glutamatergic, non‐TH, acid‐sensitive and innervate regions involved in respiratory rhythm generation. Using a lentivirus with the ChR2‐mCherry fusion protein under the PRSX8 promoter (with 8 copies of the Phox2 transcription factor response site), we drove expression of ChR2‐mCherry to Phox2b neurons with >92% selectivity. Appropriately targeted microinjections of the virus restricted transgene expression to the RTN and nearby C1 adrenergic neurons (2.7/1 non TH/TH in control rats and 5/1 in rats with selective lesions of C1 cells). Activation of the ChR2‐transfected neurons with 470 nm; 20 Hz, 10 ms pulses, 30s trains of pulsed laser light (LL) produced large increases in phrenic nerve discharge (PND), a 5mmHg increase in BP and a 10% rise in sympathetic nerve discharge (SND). PND activation decayed slowly after turning off LL (t1/2=11.8s) whereas SND activation returned to control within seconds. LL increased PND equally in control and C1‐lesion rats with no BP change in the latter. LL had no effect on PND or BP in animals with RTN transfection using control lentivirus of comparable titer. In summary, activation of RTN stimulates respiratory activity with a slow time course consistent with a chemoreceptor role, whereas C1 neuron stimulation primarily affects BP.
Stimulating opioid μ‐receptors in the commissural subdivision of the nucleus tractus solitarius (cNTS) abolishes the carotid body‐mediated ventilatory response in anesthetized rats
Authors: Zhang Zhenxiong , Zhang Cancan , Xu Fadi
Published: June 2021

Carotid chemoreceptors are essential in generating hypoxic ventilatory response (HVR) and their afferents primarily terminate in the cNTS. As the cNTS contains the highest expression of μ‐receptors in the brainstem, we tested whether activating cNTS μ‐receptors modulated the carotid body‐mediated HVR. In anesthetized and spontaneously breathing rats, cardiorespiratory activities and their responses to pure N2 administered for 10 s were recorded before and after microinjection of μ‐receptor agonist, DAMGO (≈5 nl), into the cNTS. DAMGO concentration at 0.5 and 2.5 mM caused a similar increase in blood pressure (26% and 33%) and decrease in ventilation (13% and 11%) due to the depression of VT. The transient N2‐induced ventilatory response was attenuated (64% reduction) by the low dose, and abolished by the high dose of DAMGO due to the impact on both VT and f responses. Similarly, the ventilatory responses to intra‐carotid delivery of NaCN (5 μg/50 μl) were eliminated after microinjection of the high dose of DAMGO into the cNTS. Our data suggest that cNTS opioids are capable of blocking the carotid chemoreceptor‐mediated ventilatory responses via acting on μ‐receptors that are likely located on the synapses where the second‐order neurons receive the inputs from the carotid chemoreceptors. (Supported by ALA RT‐83131‐N)
Effects of chemical lesion of Nucleus Raphe Obscurus on the thermoregulatory and ventilatory responses to hypoxia
Authors: da Silva Glauber Ferreira , Giusti Humberto , de Castro Olagide , Dias Mirela , Garcia‐Cairasco Norberto , Gargaglioni Luciane , Branco Luiz G. , Glass Mogens
Published: June 2021

The role of Nucleus Raphe Obscurus (ROb) on the hypoxic ventilatory response (HVR) and anapyrexia is not yet understood. Our objective was to study the effects of chemical lesion of ROb on ventilatory and thermoregulatory responses to hypoxia. Seven days before the experiments, male Wistar rats received a microinjection of ibotenic acid (0.2 ug/0.1 uL) or vehicle (PBS) into the ROb. The Fluoro‐Jade (FJ) histochemistry was used for staining the degenerative neurons (FJ+) in the microinjection site and surroundings, the latter named peri‐ROb group. Pulmonary ventilation (Ve, plethismography) and body temperature (data loggers) were measured followed by 1 h of hypoxic exposure (7% O2). FJ+ROb lesions decreased significantly HVR in the initial stage (from 10 to 20min) of hypoxia exposure (1136±65 mL.kg‐1.min‐1) compared to FJ+peri‐ROb (1315±63 mL.kg‐1.min‐1) and vehicle group (1326±81 mL.kg‐1.min‐1).The decrease in HVR was mediated by respiratory frequency (RF) while tidal volume (VT) did not change. There were no differences in hypoxia‐induced anapyrexia between the three groups. The results suggest that ROb neurons exert an excitatory modulation (acting via RF) playing a role in the early stages of HVR. In the present study, there are no evidences supporting the involvement of ROb in thermoregulatory responses elicited by hypoxia. Financial Support: FAPESP
Influence of 5‐HT2A receptor blockade on hypercapnic ventilatory response in arterially‐perfused adult rat
Authors: Bale Tejus A , Solomon Irene C
Published: June 2021

A number of pathologies of breathing are postulated to arise from the failure to sense or respond appropriately to elevated levels of CO2 (hypercapnia). Serotonergic raphe neurons have been identified as putative respiratory CO2 chemosensors, and accumulating evidence suggests that disturbances in the serotonergic system may underlie these breathing pathologies. However, the role of serotonin (5‐HT) signaling in the hypercapnic ventilatory response has yet to be fully characterized. To begin to address this issue, we examined the effects of 5‐HT2A receptor blockade on the phrenic nerve discharge response to elevated levels of CO2 in 5 arterially‐perfused adult rat preparations. 5‐HT2A receptors were blocked using ketanserin (KTN; 40 μM). Although perfusion with KTN increased the frequency and decreased the amplitude of basal phrenic bursts, increasing the CO2 gassing the aCSF from 5‐10% further increased burst frequency by ~26%, produced a small (~7%) increase in burst amplitude, shifted the time‐to‐peak activity to ~20% earlier in the burst, and decreased inspiratory neural network complexity. Similar effects were observed under control conditions but the magnitude of the frequency response was slightly greater and mediated only by decreases in TE during KTN perfusion. These data suggest that activation of 5‐HT2A receptors may participate in the hypercapnic ventilatory response. Supported by NS045321