bluuebunny/crossref_metadata_2025 huggingface dataset:
ESRD Patients Have Altered Pattern of Heart Rate Variability Responses to Graded Lower Body Negative Pressure
Authors: Ye Kara , Downey Ryan , Liao Peizhou , Park Jeanie
Published: November 2023

End stage renal disease (ESRD) patients have autonomic neuropathy that contributes to increased cardiovascular (CV) risk. The normal autonomic response to orthostatic stress induced by hypovolemia is to increase sympathetic nervous system (SNS) and decrease parasympathetic nervous system (PNS) activation in order to maintain arterial blood pressure. Prior studies have shown that healthy individuals have decreases in heart rate variability (HRV) in response to increased orthostatic stress induced by graded lower body negative pressure (LBNP), reflective of increased SNS and decreased PNS activation to the heart in response to central hypovolemia. The objective of this study was to determine if ESRD patients have intact HRV responses during orthostatic stress. Given that ESRD patients have autonomic neuropathy, we hypothesized that ESRD patients have lower HRV at baseline, and an inability to adjust SNS and PNS activity to the heart in response to orthostatic stress. We measured continuous beat‐to‐beat blood pressure and ECG at baseline for 10 minutes, and during orthostatic stress induced via increasing doses of LBNP (−5, −10, −15, −20, −30, and −40 mmHg) applied for 3 minutes at each dose in ESRD patients and healthy controls. HRV was quantified as: 1)the standard deviation of the normal R‐R interval (SDNN), which is reflective of total autonomic activity; 2) the root mean square of the successive differences between adjacent N‐N intervals (RMSSD), and 3) percentage of adjacent NN intervals that differ by greater than 50 msec (pNN50), which are primarily reflective of PNS activity to the heart. We observed that baseline SDNN (p=0.001), RMSSD (p=0.004), and pNN50 (p=0.016) were significantly lower in ESRD patients compared to healthy controls, suggestive of chronically high SNS and low PNS activity in ESRD patients. The slopes of change in RMSSD and pNN50 with increasing LBNP were significantly different between ESRD and controls (p=0.004 and p<0.001, respectively). Whereas healthy controls exhibited gradual decreases in RMSSD and pNN50 with increasing LBNP, signifying decreasing PNS activity to the heart during orthostatic stress, ESRD patients exhibited constant and unchanging RMSSD and pNN50 values, suggesting failure to adjust PNS activity to the heart during orthostatic stress. In conclusion, ESRD patients had low baseline HRV measurements, indicative of high SNS and low PNS activity that could contribute to cardiovascular complications in this population. Unlike controls, ESRD patients did not demonstrate a withdrawal of PNS activation to the heart during orthostatic stress. Failure to adjust autonomic responses during orthostatic stress in ESRD may contribute to orthostatic intolerance and increased CV risk in this patient population.

Support or Funding Information

Supported by NIH HL‐098744, NIH DK‐101390, VA Merit I01CX001065, and Satellite Healthcare, a not‐for‐profit renal care provider.
Hippocampal CA1 Neuronal and Sympathetic Nerve Activity in Response to Repeated Fear Conditioning in Rats
Authors: Ukita Hiroka , Yoshimoto Misa , Nagao Kana , Miki Kenju
Published: November 2023

The aim of the present study was to assess the responses of hippocampal and sympathetic nerve activity (SNA) to fear conditioning in rats. Wistar rats were chronically instrumented with a combined probe comprising multiple electrodes (100‐μm stainless steel wires) for measurement of hippocampal CA1 neuronal activity, a glass fiber for measurement of hippocampal cerebral blood flow (CBF), electrodes for measurement of renal SNA (RSNA) and lumbar SNA (LSNA), and electroencephalogram, electromyogram, and electrocardiogram, and with a catheter for measurement of systemic arterial pressure (AP). Standard fear‐conditioning trials, in which a tone was paired with a brief electrical shock (5 mA, 1s), were given to rats twice per day over 3 days. Hippocampal CA1 neuronal activity and CBF in the CA1 region increased progressively over the 3‐day fear conditioning trial, as did AP and RSNA. By contrast, heart rate (HR) and LSNA did not change on day 2, but increased on day 3. These data suggest that the repeated fear conditioning trial causes long‐term potentiation of hippocampal CA1 neuronal activity in a progressive and additive manner. Further, this potentiation was correlated quantitatively with hippocampal CA1 CBF and RSNA, but not with LSNA or HR. Thus, CA1 activation induced by repeated fear conditioning may influence RSNA, LSNA, AP, and HR in a region‐specific manner. JSPS Grant.
Sympathetic and phrenic nerve activities in the offspring of high fat diet feeding dams
Authors: Amaral Nathalia Oda , Bassi Mirian , Sa Jessica M , Barbosa Rafaela Moreira , Zoccal Daniel , Menani Jose , Colombari Eduardo , Pedrino Gustavo , Colombari Débora Simoes Almeida
Published: November 2023

It has been demonstrated that juvenile offspring (P30) of high fat diet (HFD) feeding dams presented elevated levels of arterial pressure, probably due to a higher sympathetic nerve activity (SNA) (Hypertension, 55: 76, 2010). In the present study, we evaluated baseline and baro‐/chemoreflex control of SNA and phrenic nerve (PN) activity in offspring of HFD feeding dams. We also investigated the brain regions responsible for the maintenance of the SNA in these animals. Female rats (280–300 g) were fed with standard diet (SD) or HFD from the 6 weeks before mating until the end of lactation. After weaning (P21), the offspring were fed with SD. At P28–P32, the males of the SD‐offspring (SD‐O, n=11) or HFD‐O (n=7) underwent the in situ working‐heart‐brainstem‐hypothalamus preparation to analyze SNA and PN amplitude and frequency at baseline conditions and during chemoreflex (0.05% KCN; 50 μl) or baroreflex (0.01% phenylephrine; 100 μl) activation. At the end of the experiments a pre‐collicular transection was performed and the changes in SNA and PN activity were evaluated. We observed that there was no change in baseline SNA or PN frequency and amplitude between groups. HFD‐O showed reduced sympatho‐excitatory response to KCN compared to SD‐O (52.1 ± 10.2, vs. SD‐O: 107.3 ± 15.1%; p<0.05), greater increase in PN burst amplitude (31.8 ± 10.4 vs. SD‐O: 14.3 ± 4.1%; p<0.05) and no changes in the tachypneic response. In addition, the sympathoinhibitory response to baroreflex stimulation was decreased in HFD‐O compared to SD‐O (−32 ± 5.1%, vs. SD‐O: −56 ± 3%; p < 0.05). After the pre‐collicular transection, a significant decrease in SNA was noted in SD‐O (−28,2 ± 4,9 %, vs. baseline, p < 0.05), but not in the HFD‐O group (8.3 ± 10.9%, vs. baseline; p>0.05). There was no change in PN activity after pre‐collicular transection. The data demonstrate that in HFD‐O there is an impairment of the baroreflex and chemoreflex control of SNA. Furthermore, the SNA maintenance in these offspring seems to depend mostly on the brainstem. We still need to determine whether these changes can be responsible for the higher blood pressure levels observed in the juvenile HFD‐O rats.

Support or Funding Information

CNPq (304873/2014‐4), CAPES e FAPESP (2015/23467‐7)
Does temporary inhibition of the human primary motor cortex alter resting and isometric handgrip exercise‐mediated sympathetic neural responses?
Authors: Kimmerly Derek S. , Boe Shaun , Ladouceur Michel , Weir Julie A.
Published: November 2023

Resting muscle sympathetic nerve activity (MSNA) is elevated in stroke patients with damage encompassing the primary motor cortex (M1) and during single‐pulse, inhibitory transcranial magnetic stimulation (TMS) protocols over M1 in healthy participants. These findings suggest a suppressive role of M1 on resting levels of MSNA. It is currently unclear whether inhibition of M1 alters resting and/or exercise‐mediated sympathetic activity in humans. We tested the hypothesis that temporary inhibition of the contralateral hand region of M1 would exaggerate resting and isometric handgrip exercise (IHG)‐mediated MSNA. 12 (7♀) young, healthy (23±3 years, 25±3 kg•m−2) participants were instrumented for recordings of fibular MSNA (microneurography), heart rate (HR, electrocardiography), and non‐invasive blood pressure (BP, Portapres®). These measures were collected at rest, during 2 minutes of IHG (35% maximum effort) and 2 minutes of post‐exercise ischemia (PEI). This protocol was repeated under the following theta burst TMS (TBS) conditions: Sham (no TBS), continuous TBS (cTBS, Inhibitory) and intermittent TBS (iTBS, Facilitatory). Sham was always performed first followed by randomly assigned cTBS and iTBS conditions. Compared to Sham (30±7 bursts•100 heartbeats−1), MSNA burst incidence was higher (p<0.02) five minutes following cTBS (42±13 bursts•100 heartbeats−1) but unchanged (p=0.49) after iTBS (30±12 bursts•100 heartbeats−1). Resting HR and mean arterial BP (MAP) recorded during Sham were unchanged (all, p>0.51) following iTBS and cTBS. From rest, MSNA increased (p<0.05) after IHG (38±9 bursts•minute−1) and remained elevated following PEI (37±8 bursts•minute−1) during the Sham condition. Furthermore, HR and MAP responses peaked (both, p < 0.01) after 2‐minutes of IHG but returned to resting levels following PEI. These MSNA and cardiovascular responses to IHG and PEI were not different (all, p>0.37) in the cTBS and iTBS conditions. In conclusion, temporary inhibition of the hand region in M1 via cTBS produced a transient sympathoexcitation but did not alter resting HR or MAP. Neither facilitatory (iTBS) nor inhibitory (cTBS) stimulation protocols altered IHG‐mediated sympathetic or cardiovascular reflex responses. These findings warrant future investigation aimed at determining whether cTBS protocols can reduce resting MSNA in patient populations characterized by exaggerated sympathoexcitation (e.g., resistant hypertension, ischemic stroke).

Support or Funding Information

Support provided by: Canadian Foundation for Innovation: Leaders Opportunity Fund
Identification of Stress‐Activated Inputs to the Dorsomedial Hypothalamus
Authors: Prestipino Louise , Ouro Emily A , Jayaratne Sachini K , Polson Jaimie W
Published: November 2023

Acute psychological stress elicits a typical response characterized in the short term by elevated sympathetic nerve activity and increases in heart rate and blood pressure. While the primary neural circuitry that underpins these responses is still uncertain, the dorsomedial hypothalamus (DMH) is known to play a major role.1 However, the antecedent inputs responsible for DMH activation in response to stress are unknown. In the present study, retrograde tracing was combined with Fos immunohistochemistry following air jet stress (AJS) to identify neurons that both project to the DMH and are activated by stress.

Sprague Dawley rats received a unilateral injection of 15–30 nl of retrograde tracer choleratoxin B subunit‐AlexaFluor 555 into the DMH. One week later rats were exposed to a 15 minute AJS protocol (n=4), while controls (n=2) were left undisturbed. Following perfusion, brain sections were processed for Fos immunohistochemistry and photographed. The distribution of retrogradely and/or Fos‐labeled neurons was mapped using a rat brain atlas.2 The number of labeled cells in key regions was counted. Data are expressed as mean ± SE cells per section.

Retrogradely labeled neurons were found throughout the brainstem and forebrain (Table 1). The majority of labeled cells were located in the forebrain, in particular in the medial prefrontal cortex, lateral septal nucleus, claustrum and amygdala. Following AJS, Fos‐positive neurons were located in many, but not all, of the regions in which retrogradely labeled neurons were found. There was a significant increase in the number of Fos‐positive neurons in the Kolliker Fuse nucleus (KF) in the pons, the dorsomedial, dorsolateral and ventrolateral midbrain periaqueductal gray (PAG), amygdala, the cingulate cortex, lateral septal nucleus and the claustrum. Double‐labeled neurons were found in the brainstem in the KF and ventrolateral PAG, and in the forebrain in the amygdala, cingulate cortex, lateral septal nucleus and claustrum. However, overall there were very few double‐labeled neurons seen throughout the brain.

Neurons that both project to the DMH and are activated by AJS were identified in several brain regions, particularly in the forebrain. The number of such neurons was very small, suggesting that these neurons may play a minor role in stress‐induced activation of the DMH. An alternate hypothesis is that recruitment of DMH in response to stress may be dependent upon disinhibition.

Number of labeled cells in key nuclei

Region
CTb
Fos
CTb + Fos

RVLM
0.7±0.4
9.2±5.1
0.0±0

RVMM
0.3±0.2
6.1±4
0.0±0

PBC
27.6±14.2
14.4±5.9
1.5±0.8

KF
20.2±14.8
11.0±4.0*
0.6±0.4*

dmPAG
4.4±2.1
7.3±1*
0.2±0.2

dIPAG
4.2±1.5
10.7±0.6*
0.5±0.4

IPAG
8.6±2.7
19.9±2.4
0.3±0.1

vIPAG
12.0±1.6
18.4±3.2*
1.0±0.5*

Amy
124.4±22.7
55.9±18.3*
2.4±0.7*

Cingular Cor
88.8±32.1
17.0±0.3*
3.2±0.8*

Lat SN
177.9±62.1
38.5±5.5*
4.9±1.8*

Claustrum
148.3±55.2
35.8±11*
2.1±0.4*

mPFC
192.0±79
42.4±21.1
3.3±0.8

Values are mean ± SE
The Impact of Nitrate Supplementation on the Peripheral Chemoreceptor Reflex to Hypoxia in Older Adults
Authors: Bock Josh , Limberg Jacqueline , Hughes William , Kruse Nicholas , Ueda Ken‐ichi , Casey Darren
Published: November 2023

Peripheral chemoreceptors located within the carotid bodies activate cardiorespiratory reflexes in response to hypoxia. Carotid body chemoreceptor activity has been shown to be enhanced in conditions such as congestive heart failure, hypertension, and possibly aging. Nitric oxide (NO) has been proposed as an inhibitory modulator of carotid body chemosensitivity in response to hypoxia. Additionally, NO bioavailability is often reduced with cardiovascular risk factors such as aging. In the present study, we tested the hypothesis that increasing NO bioavailability via dietary nitrate (NO3) supplementation would reduce cardiorespiratory responses to hypoxia in older adults. A double‐blind, randomized, placebo‐controlled, cross‐over study design was used to evaluate minute ventilation (VE) and heart rate (HR) responses to acute hypoxia in 7 older adults (66±2 years; 5M/2F) before and after 4 weeks of NO3 supplementation (beetroot powder) and placebo (beetroot powder devoid of NO3). On each study visit, subjects underwent two separate isocapnic hypoxic tests (self‐regulating, partial rebreathing system with 10% O2). Subjects remained on 10% O2 until SpO2 reached ≤80%. The hypoxic exposures were separated by a 15‐minute washout period with the subjects breathing room air. VE, HR, and SpO2 were continuously monitored throughout each hypoxic exposure. Data were grouped together using a 7‐breath average with the slope of the linear regression line for each dependent variable (VE and HR) versus SpO2 serving as a measure of peripheral chemosensitivity. The slopes for the two hypoxia tests on each study visit were averaged and used for statistical comparisons. On study day one (true baseline) acute hypoxia increased both VE and HR (P<0.05 for both). Four weeks of NO3 supplementation reduced the hypoxic ventilatory response (−0.16 ± −0.05 vs. −0.06 ± −0.03 L/minute/%SpO2, P<0.05), whereas no change was observed following the placebo phase of the study (−0.14 ± −0.07 vs. −0.14 ± −0.08 L/minute/%SpO2, P=0.67). Additionally, the heart rate responses to acute hypoxia were also reduced following NO3 supplementation (−0.49 ± −0.08 vs. −0.30 ± −0.05 beats/minute/%SpO2, P<0.05), but not with placebo (−0.54 ± −0.11 vs. −0.45 ± −0.08 beats/minute/%SpO2, P=0.21). Our preliminary findings suggest that supplementation of NO3 to boost NO bioavailability in older adults blunts the cardiorespiratory responses to hypoxia. Future studies should examine the therapeutic potential of NO3 in patient populations exhibiting exaggerated peripheral chemosensitivity.

Support or Funding Information

Research supported by Neogenis Inc.
Acute intermittent hypoxia (AIH) and continuous hypoxia increase augmented breaths and efferent splanchnic sympathetic nerve activity in conscious rats
Authors: Coldren Kevin Max , Hasser Eileen , Heesch Cheryl
Published: November 2023

Previously we reported an intensity dependent effect of continuous hypoxia to increase minute ventilation and augmented breaths (high amplitude spikes in plethysmography traces). Lesion of catecholaminergic neurons projecting to the PVN attenuated ventilatory responses to hypoxia, suggesting an important role for the PVN. Recent immunohistochemistry experiments revealed that pre‐sympathetic neurons in the PVN were activated by acute intermittent hypoxia (AIH, 5 bouts, 3 min 10% O2, separated by 5 min of 21% O2) but not by continuous 10% or 8% O2 (2 hr). Current experiments in chronically instrumented conscious rats evaluated the functional effects of continuous 8% O2 (2 hr, n = 5) and AIH (n = 5) on mean arterial pressure (MAP), heart rate (HR) and integrated efferent splanchnic sympathetic nerve activity (int. SSNA). Effects of AIH on diaphragm EMG were determined in a separate group of rats (n=9). Continuous 8% O2: Qualitatively similar to previous data using 10% O2, 2 hr continuous 8% O2 resulted in a sustained decrease in MAP (baseline = 112 ±3; 2 hr 8% O2 = 86 ± 2 mmHg) and an initial increase in HR from baseline (334 ± 6 beats/min) to 424 ± 26 beats/min at 5 min. Within 5 min of exposure to 8% O2, int SSNA increased (+258 ±22%) and remained elevated at 2 hrs (+179±92%). MAP, HR, and int SSNA returned towards control levels within 10 min after return to normoxia (21% O2). Acute Intermittent Hypoxia: MAP, HR, and diaphragm EMG were measured in 9 conscious rats previously instrumented with telemetry probes. AIH bouts increased MAP (e.g. AIH5= +7±1 mmHg), HR (e.g. AIH5= +83±8 beats/min), integrated diaphragm EMG amplitude (e.g. AIH5= +25±7%), and frequency of regular breathing (e.g. AIH5= +49±7 breaths/min). Similar to responses with continuous hypoxia, regularly spaced augmented breaths (high amplitude diaphragm EMG) were evident during bouts of AIH (e.g. AIH5= 3±1/min). In a separate group of chronically instrumented conscious rats (n =5), MAP, HR and SSNA were monitored during the AIH protocol. Similar to telemetry data, bouts of hypoxia increased MAP (e.g. AIH5= +8±2 mmHg) and HR (e.g. AIH5= +75±7 beats/min). In addition SSNA increased during hypoxic bouts (e.g. AIH5= +183±38 % from baseline). Interestingly, it appears that regularly spaced large bursts in SSNA occurred at a frequency (e.g. AIH5 =3±0/min) similar to the frequency of high amplitude bursts that were observed in diaphragm EMG in telemetry experiments. Combined with immunohistochemistry findings, we conclude that activation of pre‐sympathetic neurons in the PVN could possibly contribute directly to the increased SSNA during AIH, but activation of SSNA during continuous hypoxia (8% O2) appears to be mediated by pathways distinct from the PVN.

Support or Funding Information

Funded by NIH HL 98602.
Expression of adrenergic alpha 1 receptor subtypes in magnocellular, pre‐autonomic and medial parvocellular regions of the paraventricular nucleus of the hypothalamus (PVN): Effects of acute intermittent hypoxia
Authors: Heesch Cheryl Miller , Coldren Kevin Max , Barr Stacy
Published: November 2023

Catecholaminergic inputs to magnocellular, pre‐autonomic, and medial parvocellular neuroendocrine cells in the PVN may participate in activation of these neurons during chemoreflex stimulation. Current experiments evaluated the effects of acute intermittent hypoxia (AIH, 3 min 10% O2, separated by 5 min) on regional expression of alpha1 adrenergic receptor subtypes within the PVN. Spinally projecting PVN neurons were pre‐labeled in rats by prior IML injections of Fluorogold (2%,180 nl bilateral) to aid in visual identification of pre‐autonomic regions of the PVN. Conscious rats were exposed to AIH, while normoxic control rats (N) were exposed to changes in gas flow over the same times, but O2 concentration remained at 21%. One hour later, rats were deeply anesthetized, brains were removed, and the forebrains sectioned (10 μm) on a cryostat. Laser Capture Microscopy (LCM) was used to capture magnocellular, pre‐autonomic and medial parvocellular neurons in the PVN. RNA was isolated, cDNA synthesized and, using target specific primers, expression of mRNA for alpha 1 adrenergic receptor subtypes, 1a, 1b, and 1d and the reference gene GAPDH was evaluated by real time RT‐PCR. Capture of cell populations enriched for specific regions of the PVN was verified by high relative expression of mRNA for vasopressin in the magnocellular (1±0.55) compared to the medial (0.05±0.01) and pre‐autonomic regions (0.13±0.1), and high relative expression of glucocorticoid receptor mRNA in the medial (1±0.33) versus the magnocellular (0.16±0.03) and pre‐autonomic regions (0.09±0.02). Expression of the three alpha 1 receptor subtypes was not changed by AIH in pre‐autonomic and magnocellular regions of the PVN. In the medial parvocellular region, AIH decreased expression of alpha 1d receptors (N= 1±0.35; AIH = 0.28±0.06), while expression of alpha 1a and alpha 1b receptor subtypes remained unchanged. Using Fos‐IR as an index of neuronal activation, immunohistochemistry experiments found that, compared to continuous hypoxia (2 hr, 10% O2), activation of CRH‐IR neurons in the medial parvocellular region of the PVN was significant but modest in rats exposed to AIH (continuous hypoxia = 28±6%; AIH = 7±1%). It is possible that downregulation of adrenergic alpha 1d receptors in the medial parvocellular region during AIH limits excitation of these neurons.

Support or Funding Information

Funded by Univ. MO Research Board to CMH and NIH HL 98602.
Tumor Necrosis Factor Alpha Increases Excitability in Subfornical Organ Neurons
Authors: Simpson Nick J , Ferguson Alastair V
Published: November 2023

Tumor necrosis factor‐alpha (TNFα) is a pro‐inflammatory cytokine implicated in cardiovascular diseases involving hypertension. It has been shown that TNFα−/− mice do not develop angiotensin II‐induced hypertension, although the mechanism for this is unknown. Recent studies suggest that the subfornical organ (SFO) is critical for TNFα's hypertensive effects. We therefore used patch clamp techniques to examine both acute and longer term effects of TNFα's on the excitability of dissociated SFO neurons. It was found that bath application of 500 pM TNFα induced acute depolarizations in 58% (n = 11/19) of SFO neurons tested (mean depolarization of 6.7 ± 0.8 mV, n = 11). TNFα's acute effects were concentration‐dependent with depolarizing effects observed on SFO neurons at 50 pM (44%, mean depolarization of 6.8 ± 1.0 mV, n = 7/16), and 500fM (22%, mean depolarization of 3.1 ± 0.4 mV, n = 2/9). SFO neurons incubated with TNFα for 24 hours at 575 pM showed increased action potential frequency compared to control SFO neurons (3.7 ± 1.3 Hz, TNFα, n = 7, 0.4 ± 0.2 Hz, control, n = 7, p < 0.05, Student's t‐test). Additionally, TNFα incubation reduced the action potential threshold relative to control (rheobase = 5.5 ± 0.7 pA, TNFα, n = 6, rheobase = 12 ± 1.8 pA, control, n = 6, p < 0.01, Student's t‐test). Furthermore, TNFα incubation was found to increase the magnitude of transient Na+ currents (INa) compared to control (2.7 ± 0.2 nA, TNFα, n = 12, 1.7 ± 0.1 nA, control, n = 15, p < 0.001, Student's t‐test). Lastly, it was observed that TNFα incubation shifted the activation of voltage‐gated Na+ channels to a more hyperpolarized state (V1/2 = −34.5 ± 0.8 mV, TNFα, n = 12, V1/2 = −30.2 ± 1.2 mV, control, n = 11, p < 0.001, Student's t‐test). These data suggest that TNFα is increasing the acute and long‐term excitability in SFO neurons, and this long‐term effect is influenced by INa modulation.

Support or Funding Information

CIHR
Effect of Monoamine Reuptake Inhibitors on Seizures in DBA/1 Mice
Authors: Ahamed Hassan A. , Kim Yu Jaung , Bravo Eduardo , Richerson George B.
Published: November 2023

Background

DBA/1 mice suffer from audiogenic seizures that lead to wild running, clonic seizures, tonic hind‐limb extension, and eventually seizure induced respiratory arrest (S‐IRA). The use of the selective serotonin reuptake inhibitor (SSRI) fluoxetine has been shown to prevent DBA/1 mice from having S‐IRA and tonic seizures. In the present study we use fluoxetine and the more selective agent escitalopram to determine the role serotonin reuptake plays in seizure protection in DBA/1 mice.

Hypothesis

High doses of fluoxetine and escitalopram have the ability to completely protect DBA/1 mice from audiogenic seizures.

Methods

At postnatal day 21 – 24, DBA/1 mice were subjected to audiogenic seizure priming for 3 – 5 days in order to induce ~100% seizure susceptibility. After they were primed, mice were subjected to a drug dosing scheme that consisted of 0 (vehicle), 1, 3, 10, 30 or 100 mg/kg of fluoxetine in a 10% DMSO and saline solution or escitalopram in a saline solution – at the same doses. Mice were then subjected to an audiogenic stimulus (≥106 dB) 30 minutes after drug injection. The stimulus was given for 60 seconds and mice were monitored for convulsions and S‐IRA. For those mice that developed S‐IRA, a mini‐ventilator was used to resuscitate them. If a mouse survived, it was given a week for the drug to clear, then randomized to another dosing group.

Results

At 100 mg/kg of fluoxetine, DBA/1 mice were completely protected from audiogenic seizures (n = 6 of 6). These mice displayed no evidence of seizures. It was found that all other doses were unable to protect mice from seizures and S‐IRA, except for one mouse at 1 mg/kg (n = 29 of 30). Some DBA/1 mice that received 100 mg/kg as their first dose and allowed to live, developed spontaneous seizures and eventually died 4 – 6 days after injection (n = 2 of 2). At 100 mg/kg of escitalopram, there was a lower success rate relative to fluoxetine; at this dose, escitalopram completely protected some mice (n = 2 of 6), blocked tonic‐clonic seizures (n = 1 of 6), and had no effect on tonic‐clonic + S‐IRA (n = 3 of 6). At lower doses, escitalopram was completely ineffective at stopping seizures and always led to tonic‐clonic seizures + S‐IRA (n = 20 of 20).

Conclusions

DBA/1 mice were protected from audiogenic seizures at 100 mg/kg of fluoxetine. Escitalopram, does not have as strong of an anticonvulsive effect suggesting that fluoxetine may have off‐target effects that are responsible for its more powerful anticonvulsive properties. It was seen, however, that in some cases DBA/1 mice developed spontaneous seizures after exposure to a high dose of fluoxetine.

Support or Funding Information

Supported by a STRIDE fellowship from the American Physiological Society and a grant from the National Heart, Lung and Blood Institute.
Differential expression of VEGF isoforms and receptors in the phrenic motor nucleus of end‐stage SOD‐1G93A mutant rats
Authors: Satriotomo Irawan , Mitchell Gordon S.
Published: November 2023

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease causing paralysis and death from respiratory failure. Despite major loss of phrenic motor neurons (PMN) In a rodent model of familial ALS (SOD1G93A rats), breathing capacity is maintained until late in disease progression (Nichols, et al., 2013). Thus, compensatory respiratory plasticity preserves breathing capacity despite progressive respiratory motor neuron cell death. Vascular endothelial growth factor (VEGF), often regarded as an angiogenic factor, also plays a pivotal role in neuroprotection and neuroplasticity. VEGF‐A and VEGF receptor 2 (VEGFR‐2) are expressed in PMN of naïve rats, and intrathecal VEGF‐A injections induce long‐lasting phrenic motor facilitation (Dale‐Nagle et al., 2011). Little is known concerning in the expression of different VEGF isoforms or their corresponding receptors in PMN, or changes in their expression with motor neuron disease. Since VEGF may play a key role in compensating for respiratory motor neuron loss, we investigated expression of multiple VEGF isoforms (A, B, C, D, E) and receptor subtypes (VEGFR‐1/Flt‐1, VEGFR‐2/KDR/Flk‐1, VEGFR‐3) in the phrenic motor nucleus of end‐stage SOD1G93A rats (20% decrease from peak body mass). We utilized immunohistochemistry to label different VEGF isoforms and receptors within neurons (NeuN positive), astrocytes (GFAP positive) and microglia (OX‐42 positive). Presumptive phrenic motor neurons expressed VEGF‐A, with minimal or no expression of VEGF‐B, ‐C, ‐D, and ‐E. These neurons expressed all VEGF receptors (1, 2 and 3). On the other hand, reactive astrocytes expressed all VEGF isoforms and VEGF receptors 1 and 2, but not VEGFR‐3. Interestingly, activated microglia expressed only VEGF‐B, ‐C and ‐D, but not VEGF‐A or ‐E; microglia expressed VEGFR‐1 and VEGFR‐2, but not VEGFR‐3. In conclusion, multiple VEGF isoforms and receptors are differentially expressed among different cells types of the phrenic motor nucleus of SOD‐1 mutant rats. Increased VEGF expression in glial cells neighboring motor neurons may confer neuroprotection, or help surviving motor neurons compensate for the loss of their neighbors during diseases progression. However, knowledge of the specific role(s) played by different VEGF isoforms/receptors in ALS is lacking.

Support or Funding Information

Supported by McKnight Brain Institute
The Role of the Vestibular Nucleus in the Multimodal Coordination of Balance
Authors: Wittman Sam R , Miller Derek M , Yates Bill J , McCall Andrew A
Published: November 2023

Balance is a complex neurological process that makes use of multimodal sensory information. Regulation of balance relies on the integration of sensory information about the body's position and acceleration relative to gravity (vestibular inputs, provided by organs in the inner ears) with information about the body's position relative to itself (proprioceptive inputs, provided by receptors in joints and muscles). In addition, vestibular reflexes can be modified by feedback from the limbs. However, the neural populations and mechanisms underlying multimodal balance control remain to be determined. We hypothesized that neurons in the vestibular nuclei (VN) participate in multimodal balance control by integrating vestibular and proprioceptive sensory information. To test this hypothesis, we recorded activity from single VN neurons in decerebrate and conscious cats, and determined whether they responded to movement of the limb (to activate proprioceptors) and whole body (to activate vestibular receptors). Hindlimb extension‐flexion (movement of the limb in the rostral‐caudal plane) stimuli were generated via a limb‐attached motor, and vestibular stimuli were generated using a hydraulic table. In the decerebrate preparation, 70 neurons histologically demonstrated to be in the VN exhibited a response to hindlimb stimulation. These responses were classified on the basis of cell activity changes during each of four movements (midline‐to‐extension, extension‐to‐midline, midline‐to‐flexion, and flexion‐to‐midline). Most (81.4%, 57/70) of these neurons encoded information about the direction of limb movement, while the others responded similarly to movement in any direction. Of 51 of these neurons tested for a vestibular response, 56.9% (29/51) demonstrated a significant response (signal‐to‐noise ratio> 0.5). In conscious animals, vestibular stimulation was used as a search stimulus to localize VN neurons. Two‐thirds (67.7%, 65/96) of the VN cells whose activity was modulated by vestibular stimulation had a significant response to hindlimb stimulation. Only 41.5% (27/65) of these neurons encoded the direction of the leg stimulation in their responses. These results demonstrate the convergence of vestibular and somatosensory information onto individual VN neurons. However, the responses of VN neurons to hindlimb movement differed in the conscious and decerebrate preparations: responses in decerebrate preparations were much more likely to encode the direction of limb movement (p<0.001, Fisher's exact test). This indicates the presence of cortical modulation of these responses. Our future work will delve further into the role of higher brain centers in modulating the responses of VN neurons to vestibular and proprioceptive inputs.
Transient receptor potential ankyrin 1 channel in regulating emotion, learning and memory, cognition, and social behavior of mice
Authors: Lee Hsueh‐Te , Lee Kuan‐I , Lin Hui‐Ching , Lee Tzong‐Shyuan
Published: November 2023

The transient receptor potential ankyrin 1 (TRPA1) channel is a non‐selective cation channel that helps regulate inflammatory pain sensation and nociception and the development of inflammatory diseases. However, the potential role of the TRPA1 channel and the underlying mechanism in brain functions are not fully resolved. In this study, we demonstrated that genetic deletion of the TRPA1 channel in mice or pharmacological inhibition of its activity increased neurite outgrowth. In vivo study in mice provided evidence of the TRPA1 channel as a negative regulator in hippocampal functions; functional ablation of the TRPA1 channel in mice enhanced hippocampal functions, as evidenced by less anxiety‐like behavior and enhanced fear‐related or spatial learning and memory, and novel location recognition as well as social interactions. However, the TRPA1 channel appears to be a prerequisite for motor function; functional loss of the TRPA1 channel in mice led to axonal bundle fragmentation, downregulation of myelin basic protein and decreased mature oligodendrocyte population in the brain, for impaired motor function. The TRPA1 channel may play a crucial role in neuronal development and oligodendrocyte maturation and be a potential regulator in emotion, cognition, learning and memory, and social behavior.

Support or Funding Information

Ministry of Science and Technology, R.O.C.
Cellular dynamics of tissue fusion in craniofacial development and orofacial clefting
Authors: Bush Jeffrey O. , Kim Seungil , Hagan Bronwyn
Published: November 2023

Tissue fusion is a process common to the formation of multiple organs and often involves the removal of an intervening epithelial seam between two separate embryonic prominences. Tissue fusion is particularly important during craniofacial development for the proper formation of the intact upper lip and primary palate and the formation of the intact secondary palate and failure of these processes leads to cleft lip or cleft palate respectively. Whereas formation of the lip involves fusion of the medial nasal process with the lateral nasal and maxillary processes, the secondary palate forms by fusion of the secondary palatal shelves; in both cases an intervening epithelial seam must be removed for confluence of the structure. Although apoptosis is thought to be involved in tissue fusion during both processes, direct functional evidence of this is limited and the importance of apoptosis relative to other cellular behaviors is unknown. Further, the relationship between contact of the embryonic primordia and the initiation of cellular behaviors that drive palate fusion is currently unknown. Here I will discuss our recent studies utilizing mouse genetics and live imaging approaches to determine cellular mechanisms of tissue fusion during craniofacial development.

Support or Funding Information

This work was supported by grant R01DE025877 from the NIH/NIDCR.
Vimentin Regulates Activation of the NLRC4 Inflammasome
Authors: Cheng Yuan , Davis Jennifer , Lu Ziyan , Gates Khalilah , Coates Bria , Ridge Karen
Published: November 2023

NLRC4 inflammasome activation and subsequent maturation of IL‐1β has been implicated in inflammation and acute lung injury (ALI). Cellular inflammation involves two signals. The first signal is either a pathogen‐associated molecular pattern (PAMP) or damage‐associated molecular pattern (DAMP) which activates toll‐like‐receptors (TLR) which causes the NF‐κB induction of pro‐IL‐1β. The second signal, mediated by bacterial flagellin, involves formation of a NLRC4 inflammasome which activates caspase‐1 and maturation of IL‐1β. We have used a flagellin (fliC) strain of Pseudomonas aeruginosa (PAO1) to activate the NLRC4 inflammasome. Previously it was shown that vimentin expression was involved in NLRP3 inflammasome activation. We hypothesized that vimentin is important in the NLRC4 inflammatory response and that the absence of vimentin would reduce hallmarks of lung inflammation. To determine the role of vimentin in the NLRC4 inflammatory pathway, wild‐type (WT) and vimentin‐knockout (Vim−/−) mice were subjected to a lethal dose of PAO1, which induces both pro‐inflammatory cytokine expression and induces a systemic inflammatory response that is associated with significant mortality. Wild‐type mice displayed about a 2‐fold increase in mortality compared with Vim−/− mice. Vim−/− mice had a median survival of more than 40 h, whereas WT animals had a median survival of 20 h. Histopathological examination showed that the lungs of PAO1‐treated WT mice had severe alveolar damage, characterized by interstitial edema, and more fluid and debris in the airspace than the PAO1‐treated Vim−/− mice. WT mice exposed to PAO1 exhibited an increase in wet‐to‐dry lung weight ratio and total protein concentration in the bronchoalveolar lavage fluid (BALF), whereas Vim−/− mice failed to exhibit such an increase. PAO1 induces rapid production and release of pro‐inflammatory cytokines and chemokines, which are sufficient to induce lung inflammation and ALI in mice and humans. When WT and Vim−/− mice were exposed to PAO1 for 6 h, significantly more caspase‐1 and IL‐1β were found in BALF from WT mice than in Vim−/− mice. PAO1 mediated markers that are NLRC4‐independent, such as tumor necrosis factor α (TNF‐α), were not affected by the loss of vimentin. Additionally, whole lung homogenates prepared from WT and Vim−/− mice exposed to PAO1 had increased levels of pro‐IL‐1β protein. These data suggest that vimentin is not involved in the TLR‐mediated NF‐κB activation and upregulation of pro‐IL‐1β. Rather, vimentin is required for the second step in for PAO1‐induced activation of caspase‐1 and maturation of IL‐1β via the NLRC4 inflammasome.

Support or Funding Information

This work was supported by National Heart, Lung, and Blood Institute (HL128194; HL071643)
TRPV4 and Ca2+ downregulate ENaC expression and activity following a loss of barrier integrity in alveolar epithelial cells
Authors: Dagenais André , Desjardins Julie , Shabbir Waheed , Berthiaume Yves
Published: November 2023

The epithelial Na channel (ENaC) plays an essential role in lung physiology by modulating the amount of liquid lining the respiratory epithelium. This is crucial at birth for removing foetal liquid from the lung and for edema clearance during lung injury in adulthood. We reported that ENaC is expressed in alveolar epithelial cells and that pro‐inflammatory conditions such as TNFα, TGF‐β and LPS decrease its expression and function in alveolar epithelial cells in primary culture. We tested here how breaking epithelial integrity would affect ENaC expression and function. We found that mechanical wounding (MW) or disruption of tight junction by treating the cells with a 5 min EDTA treatment, induces a ~40–60% decrease of α,β and γENaC mRNA expression. ENaC short‐circuit current (Isc) recorded in Ussing chamber was decreased by 80% at 6h following MW and EDTA treatment and was correlated to αENaC mRNA expression (R=0,8379). BAPTA‐AM pre‐treatment abolishes the effect of MW or EDTA on αENaC mRNA, suggesting that [Ca2+]i is important for this modulation. Inhibition of TRPV4 a mechano‐sensitive cationic channel that drives Ca2+ in the cytoplasm with HC‐067047 (HC0) almost inhibited completely αENAC mRNA downregulation. Activation of TRPV4 with GSK‐1016790A (GSK), a specific TRPV4 agonist, downregulates αENAC mRNA and has a major impact on the Isc response of alveolar epithelial cells. The treatment elicits a rapid, but transient, Ca2+ dependent drop of the current followed by a return to the baseline that is amiloride‐insensitive. TRPV4 current activated by GSK and inhibited by HCO was also confirmed by patch clamp in cell attach configuration. By Ca2+ fluorescence microscopy with Fluo3‐AM, we found that GSK increases [Ca2+]i and that MW generates a Ca2+ wave that spreads from the margin of the scratch to the whole epithelium. Altogether our results suggest that mechanical stress induced by MW or loss of tight junction by activating TRPV4 could increase [Ca2+]i and elicit a Ca2+ wave that could affect ENaC expression and function away from the site of injury. These data are important to better understand how intercellular Ca2+ signalling could have consequence on lung liquid clearance in injured lungs.
Cell‐Free Hemoglobin Increases Lung Alveolar‐Capillary Barrier Permeability Via TLR4
Authors: Shaver Ciara M , Putz Nathan D , Bastarache Julie A , Ware Lorraine B
Published: November 2023

Objective

Elevated levels of cell‐free hemoglobin (CFH) are present in the airspace of patients with ARDS and are associated with disruption of the alveolar‐capillary barrier. Intra‐tracheal delivery of CFH is sufficient to disrupt alveolar permeability in the lung. However, the mechanisms by which this occurs are unknown. TLR4 expressed on macrophages is a key mediator of the inflammatory effects of CFH, but the role TLR4 in CFH‐induced lung epithelial injury has not been studied. We hypothesized that TLR4 would be a critical mediator of CFH‐induced alveolar‐capillary barrier permeability.

Methods

TLR4KO or wild‐type (WT) control mice were treated with intra‐tracheal ultrapure endotoxin‐free CFH (100μg). After 24 hours, bronchoalveolar lavage (BAL) was performed. Inflammation was assessed by manual cell counts with differentials and by cytokine expression (ELISA). Alveolar‐capillary barrier function was assessed by BAL protein (BCA assay). Lung epithelial injury was assessed by BAL RAGE (ELISA). For in vitro studies, MLE‐12 mouse lung epithelial cells were treated with CFH (1mg/mL) for 24 hours and paracellular permeability measured by electrical cell‐substrate impedance sensing (ECIS) in the presence or absence of TLR4 inhibitors.

Results

As compared to WT mice, TLR4KO mice had attenuated alveolar‐capillary barrier disruption in response to CFH as measured by BAL protein (476±71μg/mL vs. 291±58, p<0.001) (Figure 1). TLR4KO mice also had a 76% reduction in BAL RAGE levels (p<0.001), suggesting that TLR4 mediates lung epithelial cell injury in response to CFH. In addition, TLR4KO had markedly reduced airspace inflammation (p<0.001). To further study whether CFH had direct effects on lung epithelial cells, MLE‐12 cells were exposed to CFH in the presence or absence of TLR4 inhibitors (antibody or small molecules). Whereas CFH caused decreased resistance across the epithelial monolayer, inhibition of TLR4 binding or signaling had no effect on CFH‐mediated permeability in vitro.

Conclusions

Cell‐free hemoglobin disrupts the alveolar‐capillary barrier in the mouse lung and disrupts paracellular permeability in cultured lung epithelial cell monolayers. TLR4 is a critical mediator of lung epithelial cell injury in vivo and contributes to alveolar‐capillary barrier integrity. Inhibition of TLR4 on epithelial cells did not impact paracellular permeability in vitro. Together, these results demonstrate that CFH has both TLR4‐dependent and TLR4‐independent effects on lung epithelial cells.

Support or Funding Information

NIH HL103836, HL126671 and HL087738; Vanderbilt Faculty Search Scholars; American Thoracic Society
NADPH‐Oxidase Signaling is Dysregulated in Thromboxane Analog‐Induced Intrauterine Growth Restricted Mice Versus Appropriately Grown Mice
Authors: Robbins Mary E , Cardona Herminio J , Taylor Joann M , Perez Marta , Fung Camille , Farrow Kathryn Noel
Published: November 2023

Background

Infants with intrauterine growth restriction (IUGR) are at increased risk for bronchopulmonary dysplasia (BPD) and BPD‐associated pulmonary hypertension (PH), but this remains poorly understood. NADPH‐oxidase (Nox) have been implicated as a source of oxidative stress in both BPD and PH by others. More recently, oxidative stress has been proposed to be increased in IUGR pregnancies.

Objective

To determine if Nox signaling is disrupted in IUGR mice relative to appropriately grown mice.

Methods

Pregnant C57Bl6 mice had a micro‐osmotic pump containing U‐46619 (2000 ng/h) or vehicle (controls) implanted at E12.5 through term. U‐46619 dams had gestational hypertension. U‐46619 pups weighing ≤1.266 g at birth (<10th percentile) were considered growth restricted (GR). GR and control mice were placed in 21% O2 (room air, RA) or 75% O2 (chronic hyperoxia, CH) within 24h of birth and sacrificed at 14d. Lungs were inflation fixed and stained with hemotoxylin and eosin or harvested for tissue. At 14d, lung morphometry was assessed. Lung Nox 1, Nox2, and Nox4 expression was measured by Western blot, normalized to β‐actin.

Results

CH‐GR mice had significantly increased alveolar area and decreased alveolar counts relative to CH‐control mice (1426±219 vs. 1023±71 μm2; 26±3.4 vs. 34±2 alveoli per hpf), and RA‐GR had a trend towards increased alveolar area and significantly decreased alveolar counts relative to RA‐control mice (738±30 vs. 611±32 μm2; 48±1.5 vs. 55±2 alveoli per hpf). The RA‐GR mice had significantly less lung Nox1 and Nox4 protein expression relative to RA‐control mice, but there was no difference in lung Nox2 expression between GR and control mice in RA. Hyperoxia increased lung Nox2 and Nox4 expression in control mice, but not GR mice.

Conclusions

GR mice have disrupted lung Nox protein expression relative to control mice. GR mice have less Nox1 and Nox4 in RA and less Nox2 and Nox4 in CH. Unexpectedly, these findings suggest less oxidative stress in the GR mice even though they have more alveolar simplification both in RA and CH. The mechanism of Nox dysregulation remains to be elucidated in these mice. Others have implicated epigenetic regulatory mechanisms for the Noxs. Given aberrant promotor DNA methylation, histone modifications along the body of the gene, and nucleosome positioning have been demonstrated to prevent upregulation of other genes in our IUGR model, we speculate that these epigenetic mechanisms may also be involved in the inability to upregulate the Noxs after IUGR.

Support or Funding Information

NIH HL109478