Vehicle-injected Rats Research Articles

PO-455617-5 ANTI-ARRHYTHMIC EFFECTS OF BIOINSPIRED MODIFIED Y RNA IN RATS WITH HEART FAILURE AND PRESERVED EJECTION FRACTION

Exosomes secreted from cardiosphere-derived cells (CDCs) attenuate ventricular arrhythmias and prolong survival of rats with heart failure and preserved ejection fraction (HFpEF). Y RNA fragments were abundant in exosomes secreted from CDCs, and they mediate the anti-inflammatory and anti-fibrotic effects of CDCs. We tested the hypothesis that bioinspired modified Y RNA decrease ventricular arrhythmias in rats with HFpEF by reversing electrical remodeling. Dahl salt-sensitive (DSS) rats (n=19) were fed a high-salt diet (8% NaCl) from 7 weeks of age to induce HFpEF. Echocardiogram was performed at 14 weeks of age to confirm the development of diastolic dysfunction (increased E/E’ ratio). Symptomatic rats with diastolic dysfunction were diagnosed with HFpEF. TY1 (Therapeutic Y RNA1, a modified Y RNA in transfection reagent) was injected via the tail vein (twice weekly for 4 weeks) to rats with HFpEF (n=7). Vehicle (phosphate-buffered saline in transfection reagent) and scramble (in transfection reagent) were also injected via the tail vein as placebo (n=8 and 4, respectively). Echocardiogram and programmed electrical stimulation (PES) were performed after 4 weeks of TY1 vs. vehicle or scramble injections. Electrocardiogram was performed to measure QTc intervals as a marker for adverse electrical remodeling. Ventricular arrhythmias were induced in 80% of vehicle-injected and 100% of scramble-injected HFpEF rats by PES, as compared to only 33% of TY1-injected HFpEF rats (p=0.036). The duration of ventricular arrhythmias was significantly lower in TY1-injected HFpEF rats as compared to vehicle-injected and scramble-injected HFpEF rats (4 [IQR 0-5] beats in TY1 vs. 30 [IQR 8-54] beats in vehicle, p=0.034; vs. 21 [16-48] beats in scramble; p=0.031). QTc interval was reduced in TY1-injected HFpEF rats as compared to vehicle or scramble-injected HFpEF rats (QTc interval 238±16 ms in TY1 vs. 271±24 ms in vehicle, p=0.034; vs. 280±7 ms in scramble, p=0.017). Survival was prolonged in TY1-injected HFpEF rats as compared to vehicle or scramble-injected HFpEF rats, although statistically insignificant due to a low number of rats (86% in TY1 vs. 63% in vehicle or 75% in scramble). TY1, a modified Y RNA, reduced ventricular arrhythmias in rats with HFpEF by reversing adverse electrical remodeling of HFpEF (as manifested by a shortened QTc interval). Given these encouraging data, TY1 merits further investigation as a novel therapeutic candidate for ventricular arrhythmias.

PO-02-209 ANTI-ARRHYTHMIC EFFECTS OF BIOINSPIRED MODIFIED Y RNA IN RATS WITH HEART FAILURE AND PRESERVED EJECTION FRACTION

Exosomes secreted from cardiosphere-derived cells (CDCs) attenuate ventricular arrhythmias and prolong survival of rats with heart failure and preserved ejection fraction (HFpEF). Y RNA fragments were abundant in exosomes secreted from CDCs, and they mediate the anti-inflammatory and anti-fibrotic effects of CDCs. We tested the hypothesis that bioinspired modified Y RNA decrease ventricular arrhythmias in rats with HFpEF by reversing electrical remodeling. Dahl salt-sensitive (DSS) rats (n=19) were fed a high-salt diet (8% NaCl) from 7 weeks of age to induce HFpEF. Echocardiogram was performed at 14 weeks of age to confirm the development of diastolic dysfunction (increased E/E’ ratio). Symptomatic rats with diastolic dysfunction were diagnosed with HFpEF. TY1 (Therapeutic Y RNA1, a modified Y RNA in transfection reagent) was injected via the tail vein (twice weekly for 4 weeks) to rats with HFpEF (n=7). Vehicle (phosphate-buffered saline in transfection reagent) and scramble (in transfection reagent) were also injected via the tail vein as placebo (n=8 and 4, respectively). Echocardiogram and programmed electrical stimulation (PES) were performed after 4 weeks of TY1 vs. vehicle or scramble injections. Electrocardiogram was performed to measure QTc intervals as a marker for adverse electrical remodeling. Ventricular arrhythmias were induced in 80% of vehicle-injected and 100% of scramble-injected HFpEF rats by PES, as compared to only 33% of TY1-injected HFpEF rats (p=0.036). The duration of ventricular arrhythmias was significantly lower in TY1-injected HFpEF rats as compared to vehicle-injected and scramble-injected HFpEF rats (4 [IQR 0-5] beats in TY1 vs. 30 [IQR 8-54] beats in vehicle, p=0.034; vs. 21 [16-48] beats in scramble; p=0.031). QTc interval was reduced in TY1-injected HFpEF rats as compared to vehicle or scramble-injected HFpEF rats (QTc interval 238±16 ms in TY1 vs. 271±24 ms in vehicle, p=0.034; vs. 280±7 ms in scramble, p=0.017). Survival was prolonged in TY1-injected HFpEF rats as compared to vehicle or scramble-injected HFpEF rats, although statistically insignificant due to a low number of rats (86% in TY1 vs. 63% in vehicle or 75% in scramble). TY1, a modified Y RNA, reduced ventricular arrhythmias in rats with HFpEF by reversing adverse electrical remodeling of HFpEF (as manifested by a shortened QTc interval). Given these encouraging data, TY1 merits further investigation as a novel therapeutic candidate for ventricular arrhythmias.

Abstract 14559: Anti-Arrhythmic Effects of Exosomes Isolated From Cardiosphere-Derived Cells in Rats With Heart Failure and Preserved Ejection Fraction

Introduction: Cardiosphere-derived cells (CDCs) reverse electrical remodeling and reduce inducible ventricular arrhythmias in rats with heart failure and preserved ejection fraction (HFpEF). Exosomes isolated from CDCs (CDCexo) have anti-arrhythmic effects in various animal models but have not been tested in models of HFpEF. Hypothesis: CDCexo, when given intravenously, reverse electrical remodeling and reduce spontaneous ventricular arrhythmias in rats with HFpEF. Methods: Dahl salt-sensitive rats were fed high-salt diet (containing 8% NaCl) to induce HFpEF from 7 weeks of age. Ambulatory electrocardiogram (ECG) devices were implanted in the peritoneum at 13 weeks of age to monitor spontaneous ventricular arrhythmias. Exosomes were harvested from media conditioned by human CDCs after 48 hours of exposure to hypoxia. Echocardiogram-verified HFpEF rats (at 14 weeks of age) were injected via tail vein with human CDCexo (n=8) vs. vehicle (IMDM media, n=8) weekly for 4 weeks. ECG parameters and spontaneous development of ventricular arrhythmias were compared between the two groups. Results: QTc interval was reduced in CDCexo-injected HFpEF rats compared to vehicle-injected HFpEF rats (262±15 ms vs. 287±25 ms, p=0.0001). Two of the eight HFpEF rats injected with vehicle showed a maximal premature ventricular contraction (PVC) burden per minute of 50% while only one HFpEF rat injected with CDCexo manifested 25% maximal PVC burden per minute. Five of eight HFpEF rats injected with vehicle showed non-sustained ventricular tachycardia (VT) while only one HFpEF rat infused with CDCexo developed non-sustained VT. One rat in the vehicle group undergoing telemetry monitoring died suddenly and the cause of death was sustained VT degenerating into ventricular fibrillation (VF). The number of VT/VF episodes per rat (including non-sustained VT and sustained VT/VF) was reduced in the CDCexo group compared to the vehicle group (0.4 [0-3] vs. 13 [0-56] episodes per rat, p=0.0443). Conclusions: CDCexo recapitulated the beneficial effects of CDCs in HFpEF rats: reversing electrical remodeling and reducing ventricular arrhythmias. More importantly, an intravenous delivery route was effective for CDCexo to confer their salutary effects to rats with HFpEF.

The pericontused cortex can support function early after TBI but it remains functionally isolated from normal afferent input

Traumatically injured brain functional connectivity (FC) is altered in a region-dependent manner with some regions functionally disconnected while others are hyperconnected after experimental TBI. Remote, homotopic cortical regions become hyperexcitable after injury, and we hypothesize that this results in increased trans-hemispheric cortical inhibition, preventing reorganization of the primary injured hemisphere. Previously we have shown that temporary silencing the contralesional cortex at 1wk normalizes affected forelimb behavioral use, but not at 4wks. To investigate the potential mechanism for this and to determine whether this occurs due to restoration of afferent pathway FC, and/or reorganization of brain circuits, we probed forelimb circuit function with sensorimotor task-evoked-fMRI, resting state fMRI seed-based analysis, and exploratory structural equation modelling (SEM) of directed causal connections due to forelimb task at 1 and 4wks post-injury after temporary, contralateral silencing with intraparenchymal injection of muscimol versus vehicle, as well as from sham rats. As predicted, silencing at 1wk and 4wks post-injury decimated the contralesional cortical forelimb map evoked by stimulation of the opposite, unaffected forelimb compared to vehicle-injected injured rats indicating the success of the intervention. Surprisingly however, this also resulted in activation of the pericontused cortex ipsilateral to the stimulated forelimb at 1wk, yet this same region could not be activated by directly stimulating the opposite, injury-affected forelimb. Underpinning this were significant increases in interhemispheric FC at the level of the cortex but decreases within subcortical regions. Causal SEM analysis confirmed increased corticothalamic connectivity and suggested changes from and to bilateral thalamus are important for the effect. At 4wks post-injury only cortical increases in FC were found in response to silencing indicating a less flexible brain, and ipsilesional cortex evoked activity was mostly absent. The absence of a reinstatement of ipsilesional evoked activity through normal pathways by temporary neuromodulation despite prior data showing behavioral improvements under the same conditions, indicates that while the pericontused cortex does retain function initially after injury, it is too functionally disconnected to be controlled by normal afferent input. More significant alterations in cross-brain FC during neuromodulation at 1wk compared to 4wk post-injury, suggest that more distributed brain activity accounts for prior behavior improvements in sensorimotor function, and that hemispheric imbalance in function is causally involved in early loss of sensorimotor function in this TBI model.

Norepinephrine innervation of the supraoptic nucleus contributes to increased copeptin and dilutional hyponatremia in male rats.

Dilutional hyponatremia associated with liver cirrhosis is due to inappropriate release of arginine vasopressin (AVP). Elevated plasma AVP causes water retention resulting in a decrease in plasma osmolality. Cirrhosis, in this study caused by ligation of the common bile duct (BDL), leads to a decrease in central vascular blood volume and hypotension, stimuli for nonosmotic AVP release. The A1/A2 neurons stimulate the release of AVP from the supraoptic nucleus (SON) in response to nonosmotic stimuli. We hypothesize that the A1/A2 noradrenergic neurons support chronic release of AVP in cirrhosis leading to dilutional hyponatremia. Adult, male rats were anesthetized with 2-3% isoflurane (mixed with 95% O2/5% CO2) and injected in the SON with anti-dopamine β-hydroxylase (DBH) saporin (DSAP) or vehicle followed by either BDL or sham surgery. Plasma copeptin, osmolality, and hematocrit were measured. Brains were processed for ΔFosB, dopamine β-hydroxylase (DBH), and AVP immunohistochemistry. DSAP injection: 1) significantly reduced the number of DBH immunoreactive A1/A2 neurons (A1, P < 0.0001; A2, P = 0.0014), 2) significantly reduced the number of A1/A2 neurons immunoreactive to both DBH and ΔFosB positive neurons (A1, P = 0.0015; A2, P < 0.0001), 3) reduced the number of SON neurons immunoreactive to both AVP and ΔFosB (P < 0.0001), 4) prevented the increase in plasma copeptin observed in vehicle-injected BDL rats (P = 0.0011), and 5) normalized plasma osmolality and hematocrit (plasma osmolality, P = 0.0475; hematocrit, P = 0.0051) as compared with vehicle injection. Our data suggest that A1/A2 neurons contribute to increased plasma copeptin and hypoosmolality in male BDL rats.

Basal and Hypoxic Respiratory Behaviors in Aged 6‐OHDA‐Induced SN Lesion PD Rat Model: Effects of the 5‐HT1A Receptor Agonist Buspirone

Parkinson’s disease (PD) is progressive neurodegenerative disease, with the diagnosis of idiopathic PD occurring in humans at ~60 years of age. Most studies, however, use young adult (≤3 month old) rats to investigate PD‐related abnormalities in various physiological systems, with the most commonly used experimental rat PD model being generated by unilateral injection of the neurotoxin 6‐hydroxydopamine (6‐OHDA) into specific regions of the nigrostriatal pathway. While this model results in the loss of dopaminergic neurons and the subsequent changes in downstream neurotransmitter signaling that are characteristic of PD, it does not consider potential contributions of physical changes to the brain and neurotransmitter signaling that are part of normal aging on the progression of PD. To better understand age‐related influences and potential contributions of PD‐related abnormalities in serotonergic (5‐HT) neurotransmission on respiratory dysfunction (which is a common non‐motor symptom) in PD, we investigated basal and hypoxic (12% O2; 90s) respiratory behaviors before and after acute administration of the frequently prescribed anxiolytic 5‐HT1A receptor agonist buspirone (BUS; 0.5 mg/kg, iv) in an aged (~9 month old) female Sprague‐Dawley PD rat model produced by unilateral 6‐OHDA injection into the substantia nigra (SN). For these experiments, diaphragm EMG activity was recorded and quantified from spontaneously breathing urethane‐anesthetized rats at 2‐weeks after SN 6‐OHDA or vehicle (control) injections. We found that 6‐OHDA‐injected rats have a lower basal breathing frequency (P&lt;0.05) due to shorter expiratory duration (TE; P&lt;0.05) than vehicle‐injected rats, but both 6‐OHDA‐ and vehicle‐injected rats exhibit significant increases in breathing frequency (P≤0.001) and EMG amplitude (P≤0.023) during hypoxia; the magnitude of the peak frequency increase in 6‐OHDA‐injected rats was slightly greater (P=0.083). Both 6‐OHDA‐ and vehicle‐injected rats also showed similar post‐hypoxic frequency decline (P≤0.003), but only vehicle‐injected rats displayed a slight and sustained post‐hypoxic amplitude depression (P&lt;0.1). In vehicle‐injected rats, acute BUS injection produced a progressive decrease in basal EMG amplitude (P=0.066) and a persistent increase in breathing frequency (P=0.064) by decreasing inspiratory duration (TI; P&lt;0.001) while in 6‐OHDA‐injected rats, a transient initial increase in EMG amplitude (P=0.043) and a persistent decrease in TI (P=0.005) and TE (P=0.089) were noted. In both 6‐OHDA‐ and vehicle‐injected rats, BUS injection also slightly exaggerated the initial hypoxic frequency increase, but blunted the amplitude increase with amplitude progressively declining to near pre‐hypoxic levels before the end of the hypoxic exposure. BUS injection also shortened the duration of post‐hypoxic EMG amplitude depression in vehicle‐injected rats. These data demonstrate that some differences in basal and hypoxic respiratory behaviors exist between older 6‐OHDA‐ and vehicle‐injected rats, and that some respiratory behaviors of these rat models are differentially impacted by BUS‐induced effects on respiratory control. We suggest that this older PD rat model be further evaluated.

Stimulation of Locus Coeruleus Inhibits the Cardiogenic Sympathetic Afferent Reflex in Rats

Ischemic stimulation of cardiac sensory afferents causes chest pain and an increase in sympathetic vasomotor activity. This sympathetic reflex, which increases blood pressure and heart rate, is referred to as the cardiogenic sympathetic afferent reflex (CSAR). While the CSAR may be important for the maintenance of blood perfusion of vital organs, the increased sympathetic activity may also be detrimental because it further increases the oxygen demand of the ischemic myocardium. While the brain stem nuclei, such as the nucleus tractus solitarius and rostral ventrolateral medulla have been shown to play an important role in the CSAR, the role of other brain stem nuclei and the sources that regulate sympathetic activity in the CSAR are still not clear. We have previously characterized the role of hypothalamic nuclei in the CSAR and have shown that acute chemical inhibition of the paraventricular nucleus but not the dorsomedial hypothalamus attenuates the CSAR. However, little is known regarding other brain stem sites involved in the regulation of sympathetic activity. In this study, we tested the hypothesis that “stimulation of the locus coeruleus (LC) attenuates the CSAR in rats”. Renal sympathetic nerve activity (RSNA), blood pressure, and heart rate were recorded in anesthetized and cervically vagotomized rats during epicardial application of bradykinin (BK 10 μg/ml). Unilateral microinjection of glutamate (2.5 nmol in 50 nl), was used to stimulate the LC. Vehicle injection into the LC had no significant effect (n=10, P > 0.05) on blood pressure, heart rate, or RSNA. In vehicle-injected rats, epicardial BK application significantly increased (P < 0.05) RSNA to 253.5 ± 19.0% of baseline, heart rate from 335.7 ± 14.0 to 351.4 ± 10.5 bpm, and blood pressure from 103.4 ± 3.5 mmHg to 131.4 ± 4.1 mmHg. In a separate group of rats (n=10) glutamate microinjection into the LC significantly (P < 0.05) reduced basal RSNA to 40.2 ± 7.0% of baseline and blood pressure to 60.4 ± 5.0 mmHg, but did not significantly decrease heart rate (329.3 ± 12.5 bpm, P > 0.05). In this group of rats, although epicardial BK application significantly increased (P < 0.05) RSNA to 118.3 ± 19.3% of baseline and blood pressure to 129.5 ± 6.6 mmHg the reflex response to epicardial BK application on RSNA, blood pressure, and heart rate, was significantly attenuated (P < 0.05) compared with the respective controls. These results suggest that the brain stem pathways involving LC inputs to sympathetic vasomotor pathways are important for the regulation of the CSAR.

Repeated intrastriatal application of botulinum neurotoxin-A did not influence choline acetyltransferase-immunoreactive interneurons in hemiparkinsonian rat brain – A histological, stereological and correlational analysis

In Parkinson’s disease, dopamine depletion leads to hyperactivity of cholinergic interneurons in the caudate-putamen (CPu). Botulinum neurotoxin-A (BoNT-A) inhibits the release of acetylcholine in the peripheral nervous system and is also thought to act as a local anticholinergic drug when injected intrastriatally. In hemiparkinsonian (hemi-PD) rats, a unilateral intrastriatal injection of 1 ng BoNT-A significantly diminished apomorphine-induced rotation behavior for at least 3 months, the effect fading thereafter. A second intrastriatal BoNT-A application, 6 months after the first one, led to a stronger and longer-lasting, beneficial behavioral reaction.As a single BoNT-A injection was not cytotoxic in the rat striatum and resembled BoNT-A treatment in clinical practice, here, we investigated the structural outcome of repeated intrastriatal BoNT-A injections with respect to striatal volume, the number of choline acetyltransferase-immunoreactive (ChAT-ir) interneurons and of the length of their dendritic arbors, and the numeric density of ChAT-ir BoNT-A-induced varicosities (BiVs). Repeated unilateral intrastriatal BoNT-A application decreased the volume of the injected CPu, but did not significantly change the number of striatal ChAT-ir interneurons. Also, the total dendrite length of ChAT-ir interneurons after repeated BoNT-A application resembled the values in double vehicle-injected hemi-PD rats. In repeatedly BoNT-A-injected hemi-PD rats, the numeric density of ChAT-ir BiVs in the CPu was increased compared with rats only intrastriatally injected once with BoNT-A. Even repeated BoNT-A injections in rat striata did not cause substantial morphological changes in ChAT-ir neuron, except for the increased numeric density of ChAT-ir BiVs.

Effects of Fluoxetine on Inspiratory Motor Activity and Chemical Control of Breathing in Spontaneously Breathing Urethane‐Anesthetized 6‐OHDA MFB‐lesioned Parkinson’s Disease Rat Model

Breathing abnormalities are a common non‐motor symptom (NMS) of Parkinson’s Disease (PD), and multiple causes have been proposed, including impaired central respiratory control. While the loss of dopaminergic neurons is the primary pathology of PD, it is becoming better appreciated that serotonergic (5‐HT) dysfunction also plays a role in the development of motor and NMS and complications in PD. To this end, recent studies addressing the role of 5‐HT in PD symptoms have revealed that activation or antagonism of specific 5‐HT receptor subtypes may have therapeutic benefit. 5‐HT neurotransmission is also known to play a critical role in control of breathing; thus, respiratory abnormalities in PD may be further complicated by the progressive 5‐HT dysfunction in PD. Ongoing work in our laboratory has been focused on characterizing the respiratory phenotype of PD using 6‐hydroxydopamine (6‐OHDA) neurotoxin‐induced preclinical rat models of PD. Here, we evaluate potential contributions of 5‐HT dysfunction by assessing the effects of acute administration of the selective 5‐HT reuptake inhibitor fluoxetine hydrochloride (FLX) on basal inspiratory motor (diaphragm EMG) activity and hypoxic (HVR) and hypercapnic (HCVR) ventilatory responses in spontaneously breathing urethane‐anesthetized adult female rats 2‐weeks after unilateral medial forebrain bundle (MFB) 6‐OHDA or vehicle/control injection. Basal (BL) diaphragm EMG activity was recorded for ≥30 min, the HVR (12% O2, 90s) and HCVR (7% CO2, 5 min) were assessed, acute FLX (10 mg/kg, iv) was injected and allowed 30 min to exert steady‐state effects, and then the HVR and HCVR were re‐assessed. We found that in both vehicle‐ and 6‐OHDA‐injected rats, administration of FLX produced an initial increase in burst frequency (by ~30%) and amplitude (by ~15%) above BL levels within ~60 s, after which burst frequency attenuated to a new steady‐state level of ~10% above BL levels while burst amplitude returned to slightly below BL levels in vehicle‐injected rats, but attained a new steady‐state level of ~10% above BL levels in 6‐OHDA‐injected rats. The effects of FLX on HVR and HCVR frequency behaviors were negligible in both control‐ and 6‐OHDA‐injected rats although a slight attenuation of the HVR frequency increase (by ~5%) was seen. The effects of FLX on HVR and HCVR amplitude behaviors, while underpowered, showed a trend of slightly enhanced of HVR amplitude (by ~5%) and HCVR amplitude (by ~9%) in 6‐OHDA‐injected rats; however, additional experiments are needed to determine the significance of these observations. While these preliminary observations suggest that (compared to control rats) MFB‐lesioned rats exhibit a difference in FLX‐induced effects on basal inspiratory burst amplitude alterations, acute FLX administration may not be able to fully correct chemical control of breathing deficits. We suggest that additional experiments are needed to better identify specific 5‐HT mechanisms that may contribute to chemical control of breathing deficits in this PD rat model.Support or Funding InformationNIH NS101737; Thomas Hartman Center for Parkinson’s Disease Research at Stony Brook University

CHARACTERIZING THE ROLE OF THE DORSOMEDIAL HYPOTHALAMIC NUCLEUS IN THE CARDIOGENIC SYMPATHETIC AFFERENT REFLEX IN RATS

Myocardial ischemia causes the production and release of metabolites such as bradykinin (BK), which stimulates cardiac spinal sensory afferents causing chest pain and an increase in sympathetic activity. This sympathetic reflex, which increases heart rate and blood pressure, is referred to as the cardiogenic sympathetic afferent reflex (CSAR). During myocardial ischemia, this sympathetic reflex activity is important for the maintenance of blood pressure and perfusion of vital organs. However, the increased sympathetic activity may also be detrimental because it further increases the oxygen demand of the ischemic myocardium. While the brain stem nuclei, such as the nucleus tractus solitarius and rostral ventrolateral medulla have been shown to play an important role in the CSAR, the role of other supramedullary nuclei and the sources that generate sympathetic activity in the CSAR are still not clear. We have previously characterized the role of the paraventricular nucleus (PVN) of the hypothalamus in the CSAR and shown that acute chemical inhibition of the PVN attenuates the CSAR. However, we also have shown that the CSAR recovers in rats after chronic ibotenic acid lesion of the PVN suggesting that other supramedullary sites are involved. The dorsomedial hypothalamic nucleus (DMH) is another important hypothalamic site that plays an important role in cardiovascular sympathetic regulation. However, little is known regarding the role of the DMH in the CSAR. In this study, we tested the hypothesis that “inhibition of the DMH attenuates the CSAR in rats”. Renal sympathetic nerve activity (RSNA) and blood pressure were recorded in anesthetized rats during epicardial application of BK (10 μg/ml). Bilateral microinjection of muscimol, a GABAA agonist (0.5 nmol in 50 nl), was used to inhibit the DMH. Vehicle injection into the DMH had no significant effect (n=6, P &gt; 0.05) on blood pressure or RSNA. In vehicle‐injected rats, epicardial BK application significantly increased (P &lt; 0.05) RSNA to 204.4 ±35.08% of baseline and mean arterial pressure from 103.4 ±2.02 mmHg to 118.1 ±4.73 mmHg. In a sperate group of rats (n=8) muscimol injection into the DMH significantly (P &lt; 0.05) reduced basal blood pressure from 98.0 ±4.66 mmHg to 77.1 ±7.33 mmHg but had no significant effect (P &gt; 0.05) on baseline RSNA. In this group of rats, epicardial BK application significantly increased (P &lt; 0.05) RSNA to 193.9 ±28.00% of baseline and increased mean arterial pressure from 77.1 ±7.33 mmHg to 89.29 ±8.48 mmHg. Whereas muscimol injection into the DMH appears to attenuate the overall magnitude of the blood pressure response it had no effect on the RSNA response to epicardial bradykinin. These results suggest that in the presence of intact spinal and medullary circuits inhibition of DMH does not play an important role in the generation of sympathetic activity in the CSAR. Whereas inhibition of DMH does not appear to impair the CSAR there is good evidence to show that disinhibition of DMH increases basal vasomotor activity and augment reflex sympathetic responsiveness. Thus, our next objective is to determine if activation of DMH augments the CSAR and if so to determine the medullary connections of these DMH neurons.Support or Funding InformationNHLBI R15HL145645‐01

Enriched Environment Reverts Somatostatin Interneuron Loss in MK-801 Model of Schizophrenia.

Dysregulation of the inhibitory drive has been proposed to be a central mechanism to explain symptoms and pathophysiological hallmarks in schizophrenia. A number of recent neuroanatomical studies suggest that certain types of inhibitory cells are deficient in schizophrenia, including somatostatin-immunoreactive interneurons (SST+). The present study sought to use stereological methods to investigate whether the number of SST+ interneurons decreased after repeated injections of NMDA receptor antagonist MK-801 (0.5mg/kg) and to determine the effect of limited exposure to an enriched environment (EE) in adult life on this sub-population of inhibitory cells. Considering that somatostatin expression is highly dependent on neurotrophic support, we explored the changes in the relative expression of proteins related to brain-derived neurotrophic factor-tyrosine kinase B (BDNF-TrkB) signaling between the experimental groups. We observed that early-life MK-801 treatment significantly decreased the number of SST+ interneurons in the medial prefrontal cortex (mPFC) and the hippocampus (HPC) of adult Long Evans rats. Contrarily, short-term exposure to EE increased the number of SST+ interneurons in MK-801-injected animals, except in the CA1 region of the hippocampus, whereas this increase was not observed in vehicle-injected rats. We also found upregulated BDNF-TrkB signaling after EE that triggered an increase in the pERK/ERK ratio in mPFC and HPC, and the pAkt/Akt ratio in HPC. Thus, the present results support the notion that SST+ interneurons are markedly affected after early-life NMDAR blockade and that EE promotes SST+ interneuron expression, which is partly mediated through the BDNF-TrkB signaling pathway. These results may have important implicationsfor schizophrenia, as SST+ interneuron loss is also observed in the MK-801 pre-clinical model, and its expression can be rescued by non-pharmacological approaches.

Evidence for bidirectional and trans-synaptic parasympathetic and sympathetic propagation of alpha-synuclein in rats

The conversion of endogenous alpha-synuclein (asyn) to pathological asyn-enriched aggregates is a hallmark of Parkinson’s disease (PD). These inclusions can be detected in the central and enteric nervous system (ENS). Moreover, gastrointestinal symptoms can appear up to 20 years before the diagnosis of PD. The dual-hit hypothesis posits that pathological asyn aggregation starts in the ENS, and retrogradely spreads to the brain. In this study, we tested this hypothesis by directly injecting preformed asyn fibrils into the duodenum wall of wild-type rats and transgenic rats with excess levels of human asyn. We provide a meticulous characterization of the bacterial artificial chromosome (BAC) transgenic rat model with respect to initial propagation of pathological asyn along the parasympathetic and sympathetic pathways to the brainstem, by performing immunohistochemistry at early time points post-injection. Induced pathology was observed in all key structures along the sympathetic and parasympathetic pathways (ENS, autonomic ganglia, intermediolateral nucleus of the spinal cord (IML), heart, dorsal motor nucleus of the vagus, and locus coeruleus (LC)) and persisted for at least 4 months post-injection. In contrast, asyn propagation was not detected in wild-type rats, nor in vehicle-injected BAC rats. The presence of pathology in the IML, LC, and heart indicate trans-synaptic spread of the pathology. Additionally, the observed asyn inclusions in the stomach and heart may indicate secondary anterograde propagation after initial retrograde spreading. In summary, trans-synaptic propagation of asyn in the BAC rat model is fully compatible with the “body-first hypothesis” of PD etiopathogenesis. To our knowledge, this is the first animal model evidence of asyn propagation to the heart, and the first indication of bidirectional asyn propagation via the vagus nerve, i.e., duodenum-to-brainstem-to-stomach. The BAC rat model could be very valuable for detailed mechanistic studies of the dual-hit hypothesis, and for studies of disease modifying therapies targeting early pathology in the gastrointestinal tract.

The role of prostaglandin and E series prostaglandin receptor type 4 receptors in the development of bladder overactivity in a rat model of chemically induced prostatic inflammation.

To evaluate, using a rat model of non-bacterial prostatic inflammation, the prostaglandin production and expression profiles of E-series prostaglandin (EP) receptor subtypes, which are reportedly implicated in the development of overactive bladder, in the bladder mucosa, and to investigate the effect of EP receptor type 4 (EP4) blockade on bladder overactivity after prostatic inflammation. Male Sprague-Dawley rats were used. Prostatic inflammation was induced by formalin injection (5%; 50μL per lobe) into the bilateral ventral lobes of the prostate. At 10days after induction of prostatic inflammation or vehicle injection, bladder tissues from the deeply anaesthetized rats were harvested and separated into mucosal and detrusor layers. Then, prostaglandin E2 (PGE2) concentrations and protein levels of PGE2 receptors (EP1-4) in the bladder mucosa and detrusor were measured by ELISA and Western blotting, respectively. In separate groups of control and formalin-treated rats, awake cystometry was performed to evaluate the changes in bladder activity after prostatic inflammation. In addition, the effect of intravesical administration of a selective EP4 antagonist (ONO-AE3-208; 30μm) on bladder activity was evaluated in control rats and rats with prostatic inflammation. PGE2 concentration and protein levels of EP4, but not other EP receptor subtypes, in the bladder mucosa and detrusor layers were significantly increased in formalin-injected rats vs vehicle-injected control rats. In cystometry, rats with prostatic inflammation exhibited a significant decrease in intercontraction intervals (ICIs) compared with control rats. Intravesical application of ONO-AE3-208 (30μm), but not vehicle application, significantly increased ICIs in rats with prostatic inflammation, whereas ONO-AE3-208 at this concentration did not significantly affect any cystometric values in control rats. Because intravesical administration of an EP4 antagonist effectively improved bladder overactivity after prostatic inflammation, EP4 activation, along with increased PGE2 production in the bladder mucosa, seems to be an important contributing factor to bladder overactivity induced by prostatic inflammation. Thus, blockade of EP4 in the bladder could be a therapeutic approach to male lower urinary tract symptoms attributable to benign prostatic hyperplasia with prostatic inflammation.

Prostacyclin Analogue–Loaded Nanoparticles Attenuate Myocardial Ischemia/Reperfusion Injury in Rats

Intravenously injected ONO-1301-containing nanoparticles (ONO-1301NPs), unlike an ONO-1301 solution, selectively accumulated in the ischemia/reperfusion (I/R)-injured myocardium of rats and contributed to the prolonged retention of ONO-1301 in the targeted myocardial tissue. In the ischemic area, proangiogenic cytokines were up-regulated and inflammatory cytokines were down-regulated upon ONO-1301NP administration. Consequently, ONO-1301NP-injected rats exhibited a smaller infarct size, better-preserved capillary networks, and a better-preserved myocardial blood flow at 24 h after I/R injury, compared with those in vehicle-injected or ONO-1301 solution-injected rats. ONO-1301NPs attenuate the myocardial I/R injury via proangiogenic and anti-inflammatory effects of the drug.

Influence of Hypercapnia and Pharmacological Administration of the 5‐HT1A Receptor Agonist 8‐OH DPAT on Short‐ and Long‐Term Variability in Inspiratory Motor Activity in Two Parkinson's Disease Rat Models

Parkinson's Disease is a neurological disorder primarily caused by the loss of dopaminergic (DA) neurons, and is typically characterized by both motor symptoms that include altered motor variability and dynamics, and non‐motor symptoms (NMS). Amongst these NMS, respiratory abnormalities have been noted in PD patients and multiple causes have been proposed, including impaired central respiratory control. Dysfunction in serotonergic (5‐HT) neurotransmission is becoming better appreciated for its role in the progression of the motor and NM symptoms of PD. 5‐HT signaling is important in the control of breathing, thus dysfunction may play a role in the development of respiratory abnormalities and may contribute to alterations in breathing variability. Ongoing work in our laboratory has focused on characterizing the respiratory phenotype of PD in 6‐hydroxydopamine (6‐OHDA) neurotoxin‐induced unilateral substantia nigra (SN)‐ and medial forebrain bundle (MFB)‐lesioned rat PD models. The goals of the current study were to assess the effects of acute administration of the 5‐HT1A receptor agonist 8‐OH‐DPAT and hypercapnia independently and in combination on short‐ and long‐term breathto‐breath variability in inspiratory motor (diaphragm EMG) activity in spontaneously breathing urethane‐anesthetized adult female rats at 2‐weeks after SN (n=7) or MFB (n=12) 6‐OHDA injections; control rats received vehicle injections (SN, n=4; MFB, n=6). Using Poincaré plot analysis, we found that 6‐OHDA lesioned rats generally had greater short‐ and long‐term variability in basal burst frequency than vehicle controls. While MFB‐injected 6‐OHDA and vehicle control rats display similar amplitude variability, both groups display lower burst frequency and amplitude variability compared to SN‐injected 6‐OHDA and vehicle control rats in all conditions. During hypercapnia, short‐ and long‐term frequency and amplitude variability are inconsistently modified across all groups. Acute 8‐OH‐DPAT injection increases short‐ and long‐term frequency variability but decreases or is ineffective in altering amplitude variability in all groups of 6‐OHDA‐ and vehicle‐injected rats and exacerbates the variability associated with their hypercapnic ventilatory responses. While these preliminary observations suggest that (compared to control rats) SN‐ and MFB‐lesioned rats exhibit differences in 5‐HT1A receptor activation‐induced amplitude and frequency variability effects, additional experiments are needed to better characterize hypercapnic influences on short‐ and long‐term variability in these models and to identify specific mechanisms underlying the observed differences.Support or Funding InformationNIH NS101737; Thomas Hartman Center for Parkinson's Disease Research at Stony Brook UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Isoflurane‐Induced Hypothermia Does Not Aggravate Memory Impairment in A Sporadic Alzheimer's Disease Rat Model Produced by Intracerebroventricular Streptozotocin

Several studies have shown that hypothermia and anesthesia are risk factors for Alzheimer's disease. Most of them were performed on healthy elderly rodents. However, the effect of anesthesia‐induced hypothermia in memory in a sporadic Alzheimer's disease animal model has never been studied. In this study, Wistar rats (3‐month‐old) were induced to anesthesia with 3% isoflurane and maintained with 1.5–2% isoflurane in 100% oxygen for 3 hours. In the first 30 min of anesthesia, rats were subjected to a single bilateral intracerebroventricular injection of streptozotocin (STZ, 1 mg/kg) to produce the sporadic Alzheimer's disease model, or its vehicle (0.05 mol/L citrate buffer, pH 4.5, 2 μL/ventricle). The rats were either subjected to hypothermia or not, induced by isoflurane (hypothermic and normothermic groups, respectively). In this sense, during anesthesia, normothermic rats were kept on a heating pad, and their rectal temperature was maintained at 37.5 ± 0.5°C. Hypothermic rats were kept without a heating pad. Spatial working memory was evaluated using Morris Water Maze from the 3rd to 28th‐day postsurgery, and by Y‐maze test on the 30th‐day postsurgery. Recognition memory was evaluated on the 31st‐day postsurgery using the Novel Object Recognition Test. All the procedures were approved by and are in accordance with the local ethics committee from St Joseph's Hospital and Medical Center (protocol n° 485). In the Morris water maze test, both hypothermic and normothermic STZ‐injected rats shown similar impairment in spatial working memory compared to their respective controls, i.e., hypothermic and normothermic vehicle‐injected rats, respectively. In the Y‐maze test, normothermic STZ‐injected rats shown a deficit in spatial working memory compared to normothermic vehicle‐injected rats (p = 0.028). However, this effect was missed in hypothermic groups with no difference between STZ‐ and vehicleinjected groups (p = 0.073). Similarly, in the Novel Object Recognition Test, recognition memory was impaired in normothermic STZ‐injected rats compared to normothermic vehicleinjected rats (p = 0.012) and no effect was observed between hypothermic STZ‐ and vehicleinject rats (p = 0.118). In conclusion, isoflurane‐induced hypothermia does not aggravate the STZ‐deleterious effect in recognition memory and spacial work memory in rats.Support or Funding InformationThis work was supported by the Brazilian National Council for Scientific and Technological Development (grant 449102/2014‐9 to D.C.C.) and the São Paulo Research Foundation (grants 2015/02991‐0 to M.C.A., 2015/23426‐9 to D.C.C., and 2016/01836‐3 to R.C.L.V.).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of a histone deacetylase 3 inhibitor on extinction and reinstatement of cocaine self-administration in rats

A challenge in treating substance use disorder is that successful treatment often does not persist, resulting in relapse and continued drug seeking. One approach to persistently weaken drug-seeking behaviors is to pair exposure to drug-associated cues or behaviors with delivery of a compound that may strengthen the inhibition of the association between drug cues and behavior. We evaluated whether a selective histone deacetylase 3 (HDAC3) inhibitor could promote extinction and weaken contextual control of operant drug seeking after intravenous cocaine self-administration. Male Long-Evans rats received a systemic injection of the HDAC3 inhibitor RGFP966 either before or immediately after the first extinction session. Persistence of extinction was tested over subsequent extinction sessions, as well as tests of reinstatement that included cue-induced reinstatement, contextual renewal, and cocaine-primed reinstatement. Additional extinction sessions occurred between each reinstatement test. We also evaluated effects of RGFP966 on performance and motivation during stable fixed ratio operant responding for cocaine and during a progressive ratio of reinforcement. RGFP966 administered before the first extinction session led to significantly less responding during subsequent extinction and reinstatement tests compared to vehicle-injected rats. Follow-up studies found that these effects were not likely due to a performance deficit or a change in motivation to self-administer cocaine, as injections of RGFP966 had no effect on stable responding during a fixed or progressive ratio schedule. In addition, RGFP966 administered just after the first extinction session had no effect during early extinction and reinstatement tests, but weakened long-term responding during later extinction sessions. These results suggest that a systemic injection of a selective HDAC3 inhibitor can enhance extinction and suppress reinstatement after cocaine self-administration. The finding that behavioral and pharmacological manipulations can be combined to decrease drug seeking provides further potential for treatment by epigenetic modulation.

Acetylcholine Neurotransmitter Receptor Densities in the Striatum of Hemiparkinsonian Rats Following Botulinum Neurotoxin-A Injection.

Cholinergic neurotransmission has a pivotal function in the caudate-putamen, and is highly associated with the pathophysiology of Parkinson's disease. Here, we investigated long-term changes in the densities of the muscarinic receptor subtypes M1, M2, M3 (mAchRs) and the nicotinic receptor subtype α4β2 (nAchRs) in the striatum of the 6-OHDA-induced hemiparkinsonian (hemi-PD) rat model using quantitative in vitro receptor autoradiography. Hemi-PD rats exhibited an ipsilateral decrease in striatal mAchR densities between 6 and 16%. Moreover, a massive and constant decrease in striatal nAchR density by 57% was found. A second goal of the study was to disclose receptor-related mechanisms for the positive motor effect of intrastriatally injected Botulinum neurotoxin-A (BoNT-A) in hemi-PD rats in the apomorphine rotation test. Therefore, the effect of intrastriatally injected BoNT-A in control and hemi-PD rats on mAchR and nAchR densities was analyzed and compared to control animals or vehicle-injected hemi-PD rats. BoNT-A administration slightly reduced interhemispheric differences of mAchR and nAchR densities in hemi-PD rats. Importantly, the BoNT-A effect on striatal nAchRs significantly correlated with behavioral testing after apomorphine application. This study gives novel insights of 6-OHDA-induced effects on striatal mAchR and nAchR densities, and partly explains the therapeutic effect of BoNT-A in hemi-PD rats on a cellular level.

MP45-06 BLADDER UROTHELIAL AND AFFERENT DYSFUNCTIONS UNDERLYING BLADDER OVERACTIVITY IN RATS WITH CHEMICALLY INDUCED PROSTATIC INFLAMMATION

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research & Pathophysiology1 Apr 2018MP45-06 BLADDER UROTHELIAL AND AFFERENT DYSFUNCTIONS UNDERLYING BLADDER OVERACTIVITY IN RATS WITH CHEMICALLY INDUCED PROSTATIC INFLAMMATION Shinsuke Mizoguchi, Amanda S. Wolf-Johnson, Takahisa Suzuki, Eiichiro Takaoka, Donald B. DeFranco, Zhou Wang, Lori A. Birder, and Naoki Yoshimura Shinsuke MizoguchiShinsuke Mizoguchi More articles by this author , Amanda S. Wolf-JohnsonAmanda S. Wolf-Johnson More articles by this author , Takahisa SuzukiTakahisa Suzuki More articles by this author , Eiichiro TakaokaEiichiro Takaoka More articles by this author , Donald B. DeFrancoDonald B. DeFranco More articles by this author , Zhou WangZhou Wang More articles by this author , Lori A. BirderLori A. Birder More articles by this author , and Naoki YoshimuraNaoki Yoshimura More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.1445AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES There is increasing evidence showing the positive correlation between prostatic inflammation and lower urinary tract symptoms (LUTS) in males with benign prostatic hyperplasia (BPH). Therefore, this study used a rat model of non-bacterial prostatic inflammation to investigate the changes in bladder activity and molecular characteristics of bladder afferent pathways and urothelium after prostatic inflammation. METHODS Male SD rats were used, and prostatic inflammation was induced by formalin (5%; 50 µl per lobe) injection into bilateral ventral lobes of the prostate. Bladder function was evaluated 1 and 4 weeks after formalin injection using awake cystometry. Also, after 10 days of prostatic inflammation, prostaglandin E2 (PGE2) concentrations in bladder tissues were measured by ELISA. In bladder mucosa, protein levels of TRPV1 and PGE2 receptors (EP1 to 4) as well as mRNA levels of ASICs (1-3), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) were evaluated by Western blot and RT-PCR, respectively. NGF immunoreactivity in bladder sections was also examined. In addition, mRNA levels of TRPV1 and TRPA1 receptors were measured in bladder afferent neurons labeled by Fast Blue (FB) injected into the bladder wall using laser-capture microdissection and RT-PCR methods. RESULTS Compared to vehicle-injected rats, formalin-treated rats exhibited a significant (p<0.05) decrease in intercontraction intervals in cystometry at 1 and 4 weeks after formalin injection. Tissue inflammation evident as inflammatory cell infiltration was found in the prostate, but not in the bladder from formalin-injected rats. In rats with prostatic inflammation (10 days), bladder PGE2 concentrations were significantly increased, and protein levels of TRPV1 and EP4 receptors as well as mRNA levels of all ASICs, BDNF and NGF were significantly elevated in bladder mucosa of formalin-injected rats. NGF immunoreactivity was also increased in bladder urothelium after prostatic inflammation. In FB-labeled bladder afferent neurons, mRNA levels of TRPV1 and TRPA1 were increased compared to non-labelled neurons in prostatic inflammation rats. CONCLUSIONS These results indicate that non-bacterial inflammation confined in the prostate induces bladder overactivity, enhanced PGE2 production in the bladder and upregulations of receptors and growth factors in bladder urothelium and/or afferent pathways, which could contribute to storage LUTS in BPH patients with prostatic inflammation. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e599 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Shinsuke Mizoguchi More articles by this author Amanda S. Wolf-Johnson More articles by this author Takahisa Suzuki More articles by this author Eiichiro Takaoka More articles by this author Donald B. DeFranco More articles by this author Zhou Wang More articles by this author Lori A. Birder More articles by this author Naoki Yoshimura More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Ablation of the Right Cardiac Vagus Nerve Reduces Acetylcholine Content without Changing the Inflammatory Response during Endotoxemia.

Acetylcholine is the main transmitter of the parasympathetic vagus nerve. According to the cholinergic anti-inflammatory pathway (CAP) concept, acetylcholine has been shown to be important for signal transmission within the immune system and also for a variety of other functions throughout the organism. The spleen is thought to play an important role in regulating the CAP. In contrast, the existence of a “non-neuronal cardiac cholinergic system” that influences cardiac innervation during inflammation has been hypothesized, with recent publications introducing the heart instead of the spleen as a possible interface between the immune and nervous systems. To prove this hypothesis, we investigated whether selectively disrupting vagal stimulation of the right ventricle plays an important role in rat CAP regulation during endotoxemia. We performed a selective resection of the right cardiac branch of the Nervus vagus (VGX) with a corresponding sham resection in vehicle-injected and endotoxemic rats. Rats were injected with lipopolysaccharide (LPS, 1 mg/kg body weight, intravenously) and observed for 4 h. Intraoperative blood gas analysis was performed, and hemodynamic parameters were assessed using a left ventricular pressure-volume catheter. Rat hearts and blood were collected, and the expression and concentration of proinflammatory cytokines using quantitative reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay were measured, respectively. Four hours after injection, LPS induced a marked deterioration in rat blood gas parameters such as pH value, potassium, base excess, glucose, and lactate. The mean arterial blood pressure and the end-diastolic volume had decreased significantly. Further, significant increases in blood cholinesterases and in proinflammatory (IL-1β, IL-6, TNF-α) cytokine concentration and gene expression were obtained. Right cardiac vagus nerve resection (VGX) led to a marked decrease in heart acetylcholine concentration and an increase in cardiac acetylcholinesterase activity. Without LPS, VGX changed rat hemodynamic parameters, including heart frequency, cardiac output, and end-diastolic volume. In contrast, VGX during endotoxemia did not significantly change the concentration and expression of proinflammatory cytokines in the heart. In conclusion we demonstrate that right cardiac vagal innervation regulates cardiac acetylcholine content but neither improves nor worsens systemic inflammation.