Oxidative Stress Inhibition Via Apocynin Prevents Medullary Respiratory Neurodegeneration and Respiratory Pattern Dysfunction in a 6-Hydroxydopamine Animal Model of Parkinson's Disease.

Parkinson's Disease (PD) is a neurogenerative disorder characterized by the death of dopaminergic neurons in the Substantia Nigra pars compacta (SNpc), leading to motor, cognitive, learning, and respiratory dysfunctions. New evidence revealed that breathing impairment in PD mainly results from oxidative stress (OS) that initiates apoptotic signaling in respiratory neurons. Here, we investigated the role of OS inhibition using apocynin (non-specific NADPH oxidase inhibitor) in a 6-OHDA PD animal model in the neural control of breathing. The PD model was confirmed with a 70% reduction in TH-expressing neurons within the SNpc. After 20 and 40 days of PD induction, no differences were observed in superoxide anion levels in any respiratory nuclei. At 30 days after PD induction, 6-OHDA animals presented OS that was prevented in all respiratory nuclei by adding apocynin to the drinking water for 10 days. Forty days after PD animal model induction, impaired motor and breathing function, reduced Phox2b and NK1 receptors-expressing neurons in the medullary respiratory areas; decreased latency to fall in the rotarod motor test; and attenuated respiratory frequency and minute ventilation parameters at rest and under hypercapnia conditions were observed. After 20 days of apocynin treatment, neurodegeneration of respiratory nuclei and breathing dysfunction in 6-OHDA animals were prevented. Thus, OS contributes to respiratory neuron death, consequently leading to breathing dysfunction in the 6-OHDA PD animal model. Furthermore, these results present a new perspective for preventing the onset and progression of PD-related respiratory impairments.

Respiratory disorders of Parkinson's disease.

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra, mainly affecting people over 60 yr of age. Patients develop both classic symptoms (tremors, muscle rigidity, bradykinesia, and postural instability) and nonclassical symptoms (orthostatic hypotension, neuropsychiatric deficiency, sleep disturbances, and respiratory disorders). Thus, patients with PD can have a significantly impaired quality of life, especially when they do not have multimodality therapeutic follow-up. The respiratory alterations associated with this syndrome are the main cause of mortality in PD. They can be classified as peripheral when caused by disorders of the upper airways or muscles involved in breathing and as central when triggered by functional deficits of important neurons located in the brainstem involved in respiratory control. Currently, there is little research describing these disorders, and therefore, there is no well-established knowledge about the subject, making the treatment of patients with respiratory symptoms difficult. In this review, the history of the pathology and data about the respiratory changes in PD obtained thus far will be addressed.

Baroreflex dysfunction in Parkinson's disease: integration of central and peripheral mechanisms.

The incidence of Parkinson's disease (PD) is increasing worldwide. Although the PD hallmark is the motor impairments, nonmotor dysfunctions are now becoming more recognized. Recently, studies have suggested that baroreflex dysfunction is one of the underlying mechanisms of cardiovascular dysregulation observed in patients with PD. However, the large body of literature on baroreflex function in PD is unclear. The baroreflex system plays a major role in the autonomic, and ultimately blood pressure and heart rate, adjustments that accompany acute cardiovascular stressors on a daily basis. Therefore, impaired baroreflex function (i.e., decreased sensitivity or gain) can lead to altered neural cardiovascular responses. Since PD affects parasympathetic and sympathetic branches of the autonomic nervous system and both are orchestrated by the baroreflex system, understanding of this crucial mechanism in PD is necessary. In the present review, we summarize the potential altered central and peripheral mechanisms affecting the feedback-controlled loops that comprise the reflex arc in patients with PD. Major factors including arterial stiffness, reduced number of C1 and activation of non-C1 neurons, presence of central α-synuclein aggregation, cardiac sympathetic denervation, attenuated muscle sympathetic nerve activity, and lower norepinephrine release could compromise baroreflex function in PD. Results from patients with PD and from animal models of PD provide the reader with a clearer picture of baroreflex function in this clinical condition. By doing so, our intent is to stimulate future studies to evaluate several unanswered questions in this research area.

The retrotrapezoid nucleus and the neuromodulation of breathing.

Breathing is regulated by a host of arousal and sleep-wake state-dependent neuromodulators to maintain respiratory homeostasis. Modulators such as acetylcholine, norepinephrine, histamine, serotonin (5-HT), adenosine triphosphate (ATP), substance P, somatostatin, bombesin, orexin, and leptin can serve complementary or off-setting functions depending on the target cell type and signaling mechanisms engaged. Abnormalities in any of these modulatory mechanisms can destabilize breathing, suggesting that modulatory mechanisms are not overly redundant but rather work in concert to maintain stable respiratory output. The present review focuses on the modulation of a specific cluster of neurons located in the ventral medullary surface, named retrotrapezoid nucleus, that are activated by changes in tissue CO2/H+ and regulate several aspects of breathing, including inspiration and active expiration.

Oxidative stress in the medullary respiratory neurons contributes to respiratory dysfunction in the 6-OHDA model of Parkinson's disease.

KEY POINTS: Parkinson's disease (PD) is associated with respiratory dysfunction. In the 6-OHDA rat model of PD this is seen as a reduction in respiratory frequency and minute ventilation during normoxia and hypercapnia stimulus. Respiratory dysfunction is caused by neuronal death of medullary respiratory nuclei in the 6-OHDA model of PD. Oxidative stress can be considered a strong candidate for neurodegeneration via miR-34c downregulation and pro-apoptotic signalling in respiratory neurons, preceding the functional impairment observed in the 6-OHDA model of PD. ABSTRACT: Parkinson's disease (PD) is a neurodegenerative disease caused by dopaminergic neuron death in the substantia nigra (SN). New evidence has revealed that this neurodegeneration is the result of complex interactions between genetic abnormalities, environmental toxins, mitochondrial dysfunction and disruption of the blood-brain barrier (BBB) in the SN. In addition to classic symptoms, PD patients also exhibit respiratory failure. Here, we investigated whether oxidative stress was associated with neurodegeneration in a respiratory group (RG) of 6-OHDA-treated rats, which act as a model of PD. We analysed how oxidative stress affected apoptotic signalling in the RG 30 days after 6-OHDA treatment, shortly before commencement of breathing impairment (40 days). After 30 days, a dihydroethidium assay showed increased oxidative stress in the RG, anti-apoptotic signalling, as shown by an increase in p-Akt and BcL-2 and a decrease in Bax in the caudal aspect of the nucleus of the solitary tract (cNTS), and a decrease in p-p38 and Bax levels in the retrotrapezoid nucleus (RTN); pro-apoptotic signalling was indicated by a decrease in p-Akt and BcL-2 and an increase in Bax in the rostral ventral respiratory group (rVRG) and pre-Botzinger complex (preBotC). miR-34c, a known oxidative stress protector, was downregulated in 6-OHDA animals in the RC. After 40 days of 6-OHDA, the NTS, rVRG, preBotC and RTN exhibited reduced NeuN immunoreactivity, no BBB disruption and an increase in thiobarbituric acid reactivity. We conclude that in the 6-OHDA model of PD, oxidative stress contributes to neurodegeneration in medullary respiratory neurons.

Attenuated baroreflex in a Parkinson's disease animal model coincides with impaired activation of non-C1 neurons.

Orthostatic hypotension is one of the most common symptoms observed in Parkinson's disease (PD), a neurodegenerative disease caused by death of dopaminergic neurons in the substantia nigra pars compacta (SNc), and it is associated with denervation of the heart and impairment of the baroreflex. Here, we aimed to investigate if the impaired baroreflex was associated with lower activation of cardiovascular brainstem areas in a 6-hydroxydopamine (6-OHDA) animal model of PD. The PD model was generated with male Wistar rats by injection of 6-OHDA or vehicle into the striatum. After 20 or 60 days, the femoral vein and artery were cannulated to assess cardiovascular parameters during injection of sodium nitroprusside (SNP) or phenylephrine (Phe). Brainstem slices were submitted to immunohistochemistry and immunofluorescence. After 6-OHDA injection, 75% of the dopaminergic neurons in the SNc were absent, confirming establishment of the PD model. Intravenous (iv) injection of SNP generated reduced hypotension and tachycardia response, and the noncatecholaminergic (nonC1) neurons of the rostral ventrolateral medulla (RVLM) were less activated. Additionally, iv injection of Phe increased blood pressure and bradycardia to the same extent and activated equivalent numbers of neurons in the nucleus of the solitary tract and the caudal ventrolateral medulla as well as cholinergic neurons of the dorsal motor nucleus of the vagus and the nucleus ambiguus between control and PD animals. In summary, these data showed that in the PD model, impairment of cardiovascular autonomic control was observed only during deactivation of the baroreflex, which could be related to reduced activation of non-C1 neurons within the RVLM.

Cholinergic neurons in the pedunculopontine tegmental nucleus modulate breathing in rats by direct projections to the retrotrapezoid nucleus.

KEY POINTS: Cholinergic projections from the pedunculopontine tegmental nucleus (PPTg) to the retrotrapezoid nucleus (RTN) are considered to be important for sleep-wake state-dependent control of breathing. The RTN also receives cholinergic input from the postinspiratory complex. Stimulation of the PPTg increases respiratory output under control conditions but not when muscarinic receptors in the RTN are blocked. The data obtained in the present study support the possibility that arousal-dependent modulation of breathing involves recruitment of cholinergic projections from the PPTg to the RTN. ABSTRACT: The pedunculopontine tegmental nucleus (PPTg) in the mesopontine region has important physiological functions, including breathing control. The PPTg contains a variety of cell types, including cholinergic neurons that project to the rostral aspect of the ventrolateral medulla. In addition, cholinergic signalling in the retrotrapezoid nucleus (RTN), a region that contains neurons that regulate breathing in response to changes in CO2 /H+ , has been shown to activate chemosensitive neurons and increase inspiratory activity. The present study aimed to identify the source of cholinergic input to the RTN and determine whether cholinergic signalling in this region influences baseline breathing or the ventilatory response to CO2 in conscious male Wistar rats. Retrograde tracer Fluoro-Gold injected into the RTN labelled a subset of cholinergic PPTg neurons that presumably project directly to the chemosensitive region of the RTN. In unrestrained awake rats, unilateral injection of the glutamate (10 mm/100 nL) in the PPTg decreased tidal volume (VT ) but otherwise increased respiratory rate (fR ) and net respiratory output as indicated by an increase in ventilation (VE ). All respiratory responses elicited by PPTg stimulation were blunted by prior injection of methyl-atropine (5 mm/50-75 nL) into the RTN. These results show that stimulation of the PPTg can increase respiratory activity in part by cholinergic activation of chemosensitive elements of the RTN. Based on previous evidence that cholinergic PPTg projections may simultaneously activate expiratory output from the pFRG, we speculate that cholinergic signalling at the level of RTN region could also be involved in breathing regulation.

Interaction between the retrotrapezoid nucleus and the parafacial respiratory group to regulate active expiration and sympathetic activity in rats.

The retrotrapezoid nucleus (RTN) contains chemosensitive cells that distribute CO2-dependent excitatory drive to the respiratory network. This drive facilitates the function of the respiratory central pattern generator (rCPG) and increases sympathetic activity. It is also evidenced that during hypercapnia, the late-expiratory (late-E) oscillator in the parafacial respiratory group (pFRG) is activated and determines the emergence of active expiration. However, it remains unclear the microcircuitry responsible for the distribution of the excitatory signals to the pFRG and the rCPG in conditions of high CO2. Herein, we hypothesized that excitatory inputs from chemosensitive neurons in the RTN are necessary for the activation of late-E neurons in the pFRG. Using the decerebrated in situ rat preparation, we found that lesions of neurokinin-1 receptor-expressing neurons in the RTN region with substance P-saporin conjugate suppressed the late-E activity in abdominal nerves (AbNs) and sympathetic nerves (SNs) and attenuated the increase in phrenic nerve (PN) activity induced by hypercapnia. On the other hand, kynurenic acid (100 mM) injections in the pFRG eliminated the late-E activity in AbN and thoracic SN but did not modify PN response during hypercapnia. Iontophoretic injections of retrograde tracer into the pFRG of adult rats revealed labeled phox2b-expressing neurons within the RTN. Our findings are supported by mathematical modeling of chemosensitive and late-E populations within the RTN and pFRG regions as two separate but interacting populations in a way that the activation of the pFRG late-E neurons during hypercapnia require glutamatergic inputs from the RTN neurons that intrinsically detect changes in CO2/pH.

Orexinergic neurons are involved in the chemosensory control of breathing during the dark phase in a Parkinson's disease model.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra compacta (SNpc) and the only risk factor is aging. We showed that in 6-hydroxydopamine (6-OHDA)-model of PD there is a reduction in the neuronal profile within the brainstem ventral respiratory column with a decrease in the hypercapnic ventilatory response. Here we tested the involvement of orexin cells from the lateral hypothalamus/perifornical area (LH/PeF) on breathing in a 6-OHDA PD model. In this model of PD, there is a reduction in the total number of orexinergic neurons and in the number of orexinergic neurons that project to the RTN, without changing the number of CO2-activated orexinergic neurons during the dark phase. The ventilation at rest and in response to hypercapnia (7% CO2) was assessed in animals that received 6-OHDA or vehicle injections into the striatum and saporin anti-Orexin-B or IgG saporin into the LH/PeF during the sleep and awake states. The experiments showed a reduction of respiratory frequency (fR) at rest during the light phase in PD animals only during sleep. During the dark phase, there was an impaired fR response to hypercapnia in PD animals with depletion of orexinergic neurons in awake and sleeping rats. In conclusion, the degeneration of orexinergic neurons in this model of PD can be related to impaired chemoreceptor function in the dark phase.

The essential role of hypothalamic paraventricular nucleus nNOS in the modulation of autonomic control in exercised rats.

Nitric oxide (NO), an intercellular signaling molecule is relevant for circulatory autonomic control. Brain NO synthase (NOS) and NO levels were downregulated in pathological conditions, but rescued after exercise training. We hypothesized that exercise training was also able to improve NO modulation within the hypothalamic paraventricular nucleus (PVN) of healthy rats. Male Wistar rats were submitted to two 4-weeks protocols: i) swimming training (T) or kept sedentary (S), ii) l-arginine (62,5 mg/mL, 1 mL/day p. o.) or vehicle supplementation. Rats underwent stereotaxic surgery (PVN bilateral guide cannulas) and chronic catheterization of artery/vein. Arterial pressure (AP), heart rate (HR) and baroreflex sensitivity were recorded in conscious rats at rest and following a selective nNOS inhibitor (Nw-Propyl-l-Arginine, 4 nmol/100 nL) within the PVN. Rats were deeply anesthetized for brain perfusion/harvesting after respiratory arrest. In separate groups (T and S, l-arginine and Vehicle supplemented) not submitted to PVN cannulation, fresh and fixed brains were obtained for gene and protein nNOS expression (qPCR and immunohistochemistry) and nitrite levels (Griess reaction). T and l-arginine treatment were accompanied by resting bradycardia, augmented parasympathetic and reduced sympathetic activity to heart and vessels (power spectral analysis) and increased baroreflex sensitivity (†P < 0.05). In contrast, PVN nNOS inhibition blocked/attenuated these effects in addition to significantly increase in resting MAP and HR (with larger effects in T and l-arginine treated rats vs. respective controls, †P < 0.05). T increased nNOS gene and protein expression within the ventromedial and posterior PVN nuclei (†P < 0.05). PVN nitirite levels were also increased in T and l-arginine groups (†P < 0.05). Data strongly suggest that training by increasing NO availability within PVN preautonomic nuclei favors both the slow down of sympathetic and the augmentation of parasympathetic activity and facilitates baroreflex control, therefore improving autonomic regulation of the heart in healthy rats.

Long-term stimulation of cardiac vagal preganglionic neurons reduces blood pressure in the spontaneously hypertensive rat.

BACKGROUND: Arterial hypertension is associated with autonomic nervous system dysfunction. Different interventional strategies have been implemented in recent years for the reduction of sympathetic activity in patients with hypertension. However, the therapeutic benefit of increasing vagal tone in hypertensive patients remains largely unexplored. OBJECTIVE: Here, we describe the effects of long-term activation of vagal neural pathways on arterial pressure, heart rate arterial pressure variability and spontaneous baroreflex sensitivity in spontaneously hypertensive rats (SHR) and normotensive Wistar rats. METHODS: Brainstem vagal preganglionic neurons residing in the dorsal vagal motor nucleus (DVMN) were targeted with a lentiviral vector to induce the expression of an artificial G(s) protein-coupled receptor termed designer receptors exclusively activated by designer drugs (DREADD-Gs). The transduced neurons were activated daily by systemic administration of otherwise inert ligand clozapine-n-oxide. Arterial pressure measurements were recorded in conscious freely moving animals after 21 consecutive days of DVMN stimulation. RESULTS: Resting arterial pressure was significantly lower in SHRs expressing DREADD-Gs in the DVMN, compared with control SHRs expressing enhanced green fluorescent protein. No changes in arterial pressure were detected in Wistar rats expressing DREADD-Gs compared with rats expressing enhanced green fluorescent protein in the DVMN. Pharmacogenetic activation of DREADD-Gs-expressing DVMN neurons in SHRs was accompanied with increased baroreflex sensitivity and a paradoxical decrease in cardio-vagal components of heart rate and systolic arterial pressure variability in SHRs. CONCLUSION: These results suggest that long-term activation of vagal parasympathetic pathways is beneficial in restoring autonomic balance in an animal model of neurogenic hypertension and might be an effective therapeutic approach for the management of hypertension.

Inhibition of the hypercapnic ventilatory response by adenosine in the retrotrapezoid nucleus in awake rats.

The brain regulates breathing in response to changes in tissue CO2/H+ via a process called central chemoreception. Neurons and astrocytes in the retrotrapezoid nucleus (RTN) function as respiratory chemoreceptors. The role of astrocytes in this process appears to involve CO2/H+-dependent release of ATP to enhance activity of chemosensitive RTN neurons. Considering that in most brain regions extracellular ATP is rapidly broken down to adenosine by ectonucleotidase activity and since adenosine is a potent neuromodulator, we wondered whether adenosine signaling contributes to RTN chemoreceptor function. To explore this possibility, we pharmacologically manipulated activity of adenosine receptors in the RTN under control conditions and during inhalation of 7-10% CO2 (hypercapnia). In urethane-anesthetized or unrestrained conscious rats, bilateral injections of adenosine into the RTN blunted the hypercapnia ventilatory response. The inhibitory effect of adenosine on breathing was blunted by prior RTN injection of a broad spectrum adenosine receptor blocker (8-PT) or a selective A1-receptor blocker (DPCPX). Although RTN injections of 8PT, DPCPX or the ectonucleotidase inhibitor ARL67156 did not affected baseline breathing in either anesthetized or awake rats. We did find that RTN application of DPCPX or ARL67156 potentiated the respiratory frequency response to CO2, suggesting a portion of ATP released in the RTN during high CO2/H+ is converted to adenosine and serves to limit chemoreceptor function. These results identify adenosine as a novel purinergic regulator of RTN chemoreceptor function during hypercapnia.

Hormetic modulation of hepatic insulin sensitivity by advanced glycation end products.

Because of the paucity of information regarding metabolic effects of advanced glycation end products (AGEs) on liver, we evaluated effects of AGEs chronic administration in (1) insulin sensitivity; (2) hepatic expression of genes involved in AGEs, glucose and fat metabolism, oxidative stress and inflammation and; (3) hepatic morphology and glycogen content. Rats received intraperitoneally albumin modified (AlbAGE) or not by advanced glycation for 12 weeks. AlbAGE induced whole-body insulin resistance concomitantly with increased hepatic insulin sensitivity, evidenced by activation of AKT, inactivation of GSK3, increased hepatic glycogen content, and decreased expression of gluconeogenesis genes. Additionally there was reduction in hepatic fat content, in expression of lipogenic, pro-inflamatory and pro-oxidative genes and increase in reactive oxygen species and in nuclear expression of NRF2, a transcription factor essential to cytoprotective response. Although considered toxic, AGEs become protective when administered chronically, stimulating AKT signaling, which is involved in cellular defense and insulin sensitivity.

Cardiovascular dysfunction associated with neurodegeneration in an experimental model of Parkinson's disease.

Patients with Parkinson's disease (PD) exhibit both motor and non-motor symptoms. Among the non-motor symptoms, cardiovascular autonomic dysfunction is frequently observed. Here, we evaluated baroreflex function, vascular reactivity and neuroanatomical changes in brainstem regions involved in the neural control of circulation in the 6-hydroxydopamine (6-OHDA) model of PD. Male Wistar rats received a bilateral injection of 6-OHDA or vehicle into the striatum. After 61days, baroreflex function and vascular reactivity were assessed. The 6-OHDA and vehicle groups showed similar increases in mean arterial pressure (MAP) in response to phenylephrine (PE). However, the bradycardia observed in the vehicle group was blunted in the 6-OHDA-treated rats. Injection of sodium nitroprusside (SNP) decreased hypotension, tachycardia and vascular relaxation in 6-OHDA-treated rats. Bilateral intrastriatal 6-OHDA led to massive degeneration of tyrosine hydroxylase (TH)-immunoreactive neurons in the substantia nigra and to reductions in the numbers of A1/C1 and A5 catecholaminergic neurons while sparing A2 neurons within the nucleus of the solitary tract (NTS). 6-OHDA-treated rats also showed decreases in Phox2b-expressing neurons in the NTS and in choline acetyltransferase (ChAT) immunoreactivity in the nucleus ambiguus. Altogether, our data suggest that this model of PD includes neuroanatomical and functional changes that lead to cardiovascular impairment.

In vitro characterization of noradrenergic modulation of chemosensitive neurons in the retrotrapezoid nucleus.

Chemosensitive neurons in the retrotrapezoid nucleus (RTN) regulate breathing in response to CO2/H(+) changes and serve as an integration center for other autonomic centers, including brain stem noradrenergic neurons. Norepinephrine (NE) contributes to respiratory control and chemoreception, and, since disruption of NE signaling may contribute to several breathing disorders, we sought to characterize effects of NE on RTN chemoreception. All neurons included in this study responded similarly to CO2/H(+) but showed differential sensitivity to NE; we found that NE activated (79%), inhibited (7%), or had no effect on activity (14%) of RTN chemoreceptors. The excitatory effect of NE on RTN chemoreceptors was dose dependent, retained in the presence of neurotransmitter receptor blockers, and could be mimicked and blocked by pharmacological manipulation of α1-adrenergic receptors (ARs). In addition, NE-activation was blunted by XE991 (KCNQ channel blocker), and partially occluded the firing response to serotonin, suggesting involvement of KCNQ channels. However, in whole cell voltage clamp, activation of α1-ARs decreased outward current and conductance by what appears to be a mixed effect on multiple channels. The inhibitory effect of NE on RTN chemoreceptors was blunted by an α2-AR antagonist. A third group of RTN chemoreceptors was insensitive to NE. We also found that chemosensitive RTN astrocytes do not respond to NE with a change in voltage or by releasing ATP to enhance activity of chemosensitive neurons. These results indicate NE modulates subsets of RTN chemoreceptors by mechanisms involving α1- and α2-ARs.