Heart Matters: Cardiac Dysfunction and Other Autonomic Changes in Parkinson's Disease.

It has been more than 200 years since James Parkinson made the first descriptions of the disease that bears his name. Since then, knowledge about Parkinson's disease has been improved, and its pathophysiology, diagnosis, and treatments are well described in the scientific and medical literature. However, there is no way to prevent the disease from its progressive nature yet and only its symptoms can be minimized. It is known that the process of neurodegeneration begins before the onset of motor signs and symptoms of the disease, when diagnosis is usually made. Therefore, recognizing manifested non-motor symptoms can make an early diagnosis possible and lead to a better understanding of the disease. Autonomic dysfunctions are important non-motor manifestations of Parkinson's disease and affect the majority of patients. Importantly, heart failure is the third leading cause of death in people suffering from Parkinson's disease. Several evidences have shown the correlation between Parkinson's disease and the preexistence of cardiovascular diseases. Therefore, cardiovascular monitoring and identification of its dysfunctions can have a prodromal role for Parkinson's disease. This review presents studies of the literature that can lead to a better understanding of Parkinson's disease with special attention to its relation to heart and cardiovascular parameters.

Using RNA Interference for Purinoceptor Knockdown In Vivo.

RNA interference (RNAi) is a powerful post-transcriptional gene silencing (PTGS) induced by small double-stranded RNA (dsRNA). The method allows silencing of genes of interest by translation inhibition or by mRNA degradation. In this chapter, we provide a brief overview of the mechanisms involved in each step of gene silencing. A nonviral infusion of short siRNA into ventricular system of rats was used to study purinoceptor in the rat brain.

Bone marrow-derived mesenchymal stem cells versus adipose-derived mesenchymal stem cells for peripheral nerve regeneration.

Studies have confirmed that bone marrow-derived mesenchymal stem cells (MSCs) can be used for treatment of several nervous system diseases. However, isolation of bone marrow-derived MSCs (BMSCs) is an invasive and painful process and the yield is very low. Therefore, there is a need to search for other alterative stem cell sources. Adipose-derived MSCs (ADSCs) have phenotypic and gene expression profiles similar to those of BMSCs. The production of ADSCs is greater than that of BMSCs, and ADSCs proliferate faster than BMSCs. To compare the effects of venous grafts containing BMSCs or ADSCs on sciatic nerve injury, in this study, rats were randomly divided into four groups: sham (only sciatic nerve exposed), Matrigel (MG; sciatic nerve injury + intravenous transplantation of MG vehicle), ADSCs (sciatic nerve injury + intravenous MG containing ADSCs), and BMSCs (sciatic nerve injury + intravenous MG containing BMSCs) groups. Sciatic functional index was calculated to evaluate the function of injured sciatic nerve. Morphologic characteristics of nerves distal to the lesion were observed by toluidine blue staining. Spinal motor neurons labeled with Fluoro-Gold were quantitatively assessed. Compared with sham-operated rats, sciatic functional index was lower, the density of small-diameter fibers was significantly increased, and the number of motor neurons significantly decreased in rats with sciatic nerve injury. Neither ADSCs nor BMSCs significantly improved the sciatic nerve function of rats with sciatic nerve injury, increased fiber density, fiber diameters, axonal diameters, myelin sheath thickness, and G ratios (axonal diameter/fiber diameter ratios) in the sciatic nerve distal to the lesion site. There was no significant difference in the number of spinal motor neurons among ADSCs, BMSCs and MG groups. These results suggest that neither BMSCs nor ADSCs provide satisfactory results for peripheral nerve repair when using MG as the conductor for engraftment.

Silencing of P2X7R by RNA interference in the hippocampus can attenuate morphological and behavioral impact of pilocarpine-induced epilepsy.

Cell signaling mediated by P2X7 receptors (P2X7R) has been suggested to be involved in epileptogenesis, via modulation of intracellular calcium levels, excitotoxicity, activation of inflammatory cascades, and cell death, among other mechanisms. These processes have been described to be involved in pilocarpine-induced status epilepticus (SE) and contribute to hyperexcitability, resulting in spontaneous and recurrent seizures. Here, we aimed to investigate the role of P2X7R in epileptogenesis in vivo using RNA interference (RNAi) to inhibit the expression of this receptor. Small interfering RNA (siRNA) targeting P2X7R mRNA was injected into the lateral ventricles (icv) 6 h after SE. Four groups were studied: Saline-Vehicle, Saline-siRNA, Pilo-Vehicle, and Pilo-siRNA. P2X7R was quantified by western blotting and neuronal death assessed by Fluoro-Jade B histochemistry. The hippocampal volume (edema) was determined 48 h following RNAi. Behavioral parameters as latency to the appearance of spontaneous seizures and the number of seizures were determined until 60 days after the SE onset. The Saline-siRNA and Pilo-siRNA groups showed a 43 and 37% reduction, respectively, in P2X7R protein levels compared to respective vehicle groups. Neuroprotection was observed in CA1 and CA3 of the Pilo-siRNA group compared to Pilo-Vehicle. P2X7R silencing in pilocarpine group reversed the increase in the edema detected in the hilus, suprapyramidal dentate gyrus, CA1, and CA3; reduced mortality rate following SE; increased the time to onset of spontaneous seizure; and reduced the number of seizures, when compared to the Pilo-Vehicle group. Therefore, our data highlights the potential of P2X7R as a therapeutic target for the adjunct treatment of epilepsy.

Maternal seizures can affect the brain developing of offspring.

To elucidate the impact of maternal seizures in the developing rat brain, pregnant Wistar rats were subjected to the pilocarpine-induced seizures and pups from different litters were studied at different ages. In the first 24 h of life, blood glucose and blood gases were analyzed. (14)C-leucine [(14)C-Leu] incorporation was used to analyze protein synthesis at PN1, and Western Blot method was used to analyze protein levels of Bax, Bcl-2 and Poly(ADP-ribose) polymerase-1 (PARP-1) in the hippocampus (PN3-PN21). During the first 22 days of postnatal life, body weight gain, length, skull measures, tooth eruption, eye opening and righting reflex have been assessed. Pups from naive mothers were used as controls. Experimental pups showed a compensated metabolic acidosis and hyperglycemia. At PN1, the [(14)C-Leu] incorporation into different studied areas of experimental pups was lower than in the control pups. During development, the protein levels of Bax, Bcl-2 and PARP-1 in the hippocampus of experimental pups were altered when compared with control pups. A decreased level of pro- and anti-apoptotic proteins was verified in the early postnatal age (PN3), and an increased level of pro-apoptotic proteins concomitant with a reduced level of anti-apoptotic protein was observed at the later stages of the development (PN21). Experimental pups had a delay in postnatal growth and development beyond disturb in protein synthesis and some protein expression during development. These changes can be result from hormonal alterations linked to stress and/or hypoxic events caused by maternal epileptic seizures during pregnancy.

Variations of ATP and its metabolites in the hippocampus of rats subjected to pilocarpine-induced temporal lobe epilepsy.

Although purinergic receptor activity has lately been associated with epilepsy, little is known about the exact role of purines in epileptogenesis. We have used a rat model of temporal lobe epilepsy induced by pilocarpine to study the dynamics of purine metabolism in the hippocampus during different times of status epilepticus (SE) and the chronic phase. Concentrations of adenosine 5'-triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine in normal and epileptic rat hippocampus were determined by microdialysis in combination with high-performance liquid chromatography (HPLC). Extracellular ATP concentrations did not vary along 4 h of SE onset. However, AMP concentration was elevated during the second hour, whereas ADP and adenosine concentrations augmented during the third and fourth hour following SE. During chronic phase, extracellular ATP, ADP, AMP, and adenosine concentrations decreased, although these levels again increased significantly during spontaneous seizures. These results suggest that the increased turnover of ATP during the acute period is a compensatory mechanism able to reduce the excitatory role of ATP. Increased adenosine levels following 4 h of SE may contribute to block seizures. On the other hand, the reduction of purine levels in the hippocampus of chronic epileptic rats may result from metabolic changes and be part of the mechanisms involved in the onset of spontaneous seizures. This work provides further insights into purinergic signaling during establishment and chronic phase of epilepsy.

Neuroprotective effects of peroxisome proliferator-activated receptor alpha and gamma agonists in model of parkinsonism induced by intranigral 1-methyl-4-phenyl-1,2,3,6-tetrahyropyridine.

A large body of evidence suggests that peroxisome proliferator-activated receptor (PPAR) agonists may improve some of the pathological features of Parkinson's disease (PD). In the present study, we evaluated the effects of the PPAR-α agonist fenofibrate (100mg/kg) and PPAR-γ agonist pioglitazone (30mg/kg) in a rat model of parkinsonism induced by intranigral 1-methyl-4-phenyl-1,2,3,6-tetrahyropyridine (MPTP). Male Wistar rats were pretreated with both drugs for 5 days and received an infusion of MPTP. The experiments were divided into two parts. First, 1, 7, 14, and 21 days after surgery, the animals were submitted to the open field test. On days 21 and 22, the rats were subjected to the forced swim test and two-way active avoidance task. In the second part of the study, 24h after neurotoxin administration, immunohistochemistry was performed to assess tyrosine hydroxylase activity. The levels of dopamine and its metabolites in the striatum were determined using high-performance liquid chromatography, and fluorescence detection was used to assess caspase-3 activation in the substantia nigra pars compacta (SNpc). Both fenofibrate as pioglitazone protected against hypolocomotion, depressive-like behavior, impairment of learning and memory, and dopaminergic neurodegeneration caused by MPTP, with dopaminergic neuron loss of approximately 33%. Fenofibrate and pioglitazone also protected against the increased activation of caspase-3, an effector enzyme of the apoptosis cascade that is considered one of the pathological features of PD. Thus, PPAR agonists may contribute to therapeutic strategies in PD.

Neuroglobin is up-regulated in the cerebellum of pups exposed to maternal epileptic seizures.

To evaluate a potential insult in the cerebellum of pups exposed to maternal epileptic seizures during intrauterine life, female rats were subjected to pilocarpine-induced epilepsy. Pups from different litters were sacrificed at 1, 3, 7 and 14 post-natal days (PN) and neuroglobin (Ngb) and gliosis were analyzed in the cerebellum by Western blotting (WB) and RT-PCR. (14)C-l-leucine-[(14)C-Leu] incorporation was used to analyze protein synthesis at PN1. Nitric Oxide (NO) and thiobarbituric acid-reactive substances (TBARS) levels were also measured. Pups from naive mothers were used as controls. The mRNA level of Ngb was increased in experimental animals at PN1 ((**)p ≤ 0.001) and PN3 ((**)p ≤ 0.001), at PN7 ((***)p ≤ 0.0001) and at PN14 ((**)p ≤ 0.001) compared to the respective controls. The protein level of Ngb increased significantly in the experimental pups at PN1 ((*)p ≤ 0.05) and at PN3 ((**)p ≤ 0.001), when compared to the control pups at PN1 and PN3. At PN7 and PN14 no difference was found. The mRNA level of GFAP increased significantly about two times at PN3 ((*)p ≤ 0.05) and PN7 ((*)p ≤ 0.05) in the experimental pups when compared to the respective controls, but was unchanged in the other studied ages. Data showed that experimental pups at PN1 exhibited reduced (about 2 times, (*)p ≤ 0.05) total protein synthesis in the cerebellum when compared to control. No differences were found in the NO and TBARS levels. Our data support the hypothesis that an up-regulation of Ngb could be a compensatory mechanism in response to the hypoxic-ischemic insults caused by seizures in pups during intrauterine life.

Acute and chronic exercise modulates the expression of MOR opioid receptors in the hippocampal formation of rats.

Exercise stimulates the release of beta-endorphin and other endogenous opioid peptides that are believed to be responsible for changes in mood, perception of pain and also performance. Although the vast majority of literature data support the role of physical exercise in increasing beta-endorphin levels, indirect measures such as increased endorphin levels in peripheral blood do not reflect opioid levels in the central nervous system. The purpose of the present study was to verify whether acute and chronic exercise using both voluntary and forced exercise procedures could modify the expression of µ-opioid receptors (MOR) in rat hippocampal formation. Immunoblotting analysis showed significantly enhanced MOR expression in the hippocampal formation in the acute (forced and voluntary) exercise groups when compared to the control group. Conversely, a significant reduction of MOR expression was noted in the chronic forced and chronic voluntary exercise groups compared to the acute forced and voluntary groups respectively. MOR expression was not significantly different in rats trained using both acute or chronic exercise. Immunohistochemistry analysis showed a higher number of MOR-positive cells for acute forced and voluntary exercise groups in the CA1, CA3, hilus and dentate gyrus regions compared to the control group. Our findings indicate that acute and chronic exercise modulates MOR expression in the hippocampal formation of rats.

Physical exercise during the adolescent period of life increases hippocampal parvalbumin expression.

In order to investigate whether physical exercise during development would promote changes the calcium-binding protein parvalbumin (PV) expression in the hippocampal formation, we performed an immunostaining study after an aerobic exercise program in rats during adolescent period of life. Wistar rats were submitted to daily exercise program in a treadmill between postnatal day 21 and 60. Running time and speed were gradually increased during the subsequent days until 18m/min for 60min. After the aerobic exercise program, animals of all groups were killed and PV immunostaining procedures were performed. The results showed significant increase of protein level in the hippocampal formation and PV-immunoreactive neurons in CA1 and CA2/CA3 regions of rats submitted to exercise when compared with control rats. This finding indicates that aerobic exercise program during adolescent period promotes neuroplastic changes in hippocampal formation.

The Na+/K+ATPase activity is increased in the hippocampus after multiple status epilepticus induced by pilocarpine in developing rats.

The effects of repetitive pilocarpine-induced status epilepticus (SE) in the hippocampal Na(+)/K(+)ATPase activity were studied in developing rat. Na(+)/K(+)ATPase is a membrane-bound enzyme responsible for the active transport of sodium and potassium ions through the membrane. It is necessary to maintain neuronal excitability. The malfunction of this enzyme has been associated with neuronal hyperexcitability. The pilocarpine-induced status epilepticus in developing rats leads to neuronal hyperexcitability and brain damage. We examined the activity of the Na(+)/K(+)ATPase enzyme in hippocampus of rats submitted to 1 episode of status epilepticus on postnatal day 9 and to 3 episodes of pilocarpine-induced status epilepticus on postnatal days 7, 8 and 9. Our findings showed that one status epilepticus episode does not modify the Na(+)/K(+)ATPase activity in hippocampus of rats studied 7 or 30 days later (at P16 or P39). However, an increase in the Na(+)/K(+)ATPase activity was detected in hippocampus of rats submitted to three consecutive status epilepticus during the development studied 7 (+142%) and 30 (+400%) days following the injections. In addition, a significant reduction in the Na(+)/K(+)ATPase activity was observed in control rats at P39 compared to P16. Our data suggest that multiple pilocarpine-induced status epilepticus in developing rats induce long-lasting increase in the Na(+)/K(+)ATPase activity in the hippocampus, reflecting hyperexcitability.

Pro-epileptic effect of alfentanil in rats subjected to pilocarpine-induced chronic epilepsy.

Pharmacological induction of epileptiform activity is a complementary method to study the epileptogenic area in drug-resistant epileptic patients. Among the different activation methods, fentanyl derivatives (e.g. alfentanil) provide one of the most efficient tools in triggering epileptiform abnormalities in surgical candidates. In this study, we tested the pro-epileptic effect of different concentrations of alfentanil in hippocampal slices obtained from control and pilocarpine-treated chronic epileptic rats. The pro-convulsant action of alfentanil was also studied in control and pilocarpine-treated epileptic rats implanted with subdural and hippocampal electrodes for electroencephalographic recordings. In 90% of slices from control animals, application of alfentanil (0.1-5 microM) induced a significant enhancement in amplitude and number of population spikes recorded in the hippocampal CA1 region. In contrast, alfentanil produced a significant reduction in the amplitude of population spikes in slices from pilocarpine-treated epileptic rats. These changes were accompanied by a significant increase in the number of population spikes in the form of epileptiform multispike responses of epileptic slices. Naloxone (20 microM) antagonized the effect of alfentanil in both control and epileptic slices, reducing the number of population spikes in slices from epileptic rats. In control rats, alfentanil induced epileptiform abnormalities in the hippocampal and cortical electroencephalographic recordings but only at concentrations higher than 200 microg/kg (e.g. 350 microg/kg). Lower doses of alfentanil (25 microg/kg) elicited epileptiform abnormalities only in chronic epileptic rats. The potent action of a minimal dose of alfentanil in inducing epileptiform activity suggests an enhancement of the pro-convulsant action of mu-receptor opioids in chronic temporal lobe epilepsy.

Modulation of seizures and synaptic plasticity by adenosinergic receptors in an experimental model of temporal lobe epilepsy induced by pilocarpine in rats.

PURPOSE: Adenosine is a major negative neuromodulator of synaptic activity in the central nervous system and can exert anticonvulsant and neuroprotective effects in many experimental models of epilepsy. Extracellular adenosine can be formed by a membrane-anchored enzyme ecto-5'-nucleotidase. The purposes of this study were to characterize the role of adenosine receptors in modulating status epilepticus (SE) induced by pilocarpine and evaluate its neuroprotective action. Ecto-5'-nucleotidase activity was studied during the different phases of pilocarpine-induced epilepsy in rats. METHODS: Adult rats were pretreated with different adenosinergic agents to evaluate the latency and incidence of SE induced by pilocarpine in rats. The neuroprotective effect also was evaluated. RESULTS: A proconvulsant effect was observed with DPCPX and DMPX that reduced the latency of SE in almost all rats. Pretreatment with the MRS 1220 did not alter the incidence of SE but reduced the latency to develop SE. An anticonvulsant and neuroprotective effect was detected with R-PIA. Rats pretreated with R-PIA had a decreased number of apoptotic cells in the hippocampus, whereas pretreatment with DPCPX did not modify the hippocampal damage. An intensification of neuronal death was observed in the dentate gyrus and CA3 when rats were pretreated with DMPX. MRS-1220 did not modify the number of apoptotic cells in the hippocampus. An increase in the ecto-5 -nucleotidase staining was detected in the hippocampus during silent and chronic phases. CONCLUSIONS: The present data show that adenosine released during pilocarpine-induced SE via A1-receptor stimulation can exhibit neuroprotective and anticonvulsant roles. Similar effects could also be inferred with A2a and A3 adenosinergic agents, but further experiments are necessary to confirm their roles. Ecto-5 -nucleotidase activity during silent and chronic phases might have a role in blocking spontaneous seizures by production of inhibitory neuromodulator adenosine, besides taking part in the mechanism that controls sprouting.