Inhibition of cPLA(2) and sPLA(2) Activities in Primary Cultures of Rat Cortical Neurons by Centella asiatica Water Extract

Leaf extract of Centella asiatica has been used as an alternative medicine for memory improvement in the Indian Ayurvedic system of medicine for a long time. Although several studies have revealed its effect in ameliorating the cognitive impairment in rat models of Alzheimer's disease, the molecular mechanism of C. asiatica on neuroprotection still remains unexplained. In this study, we investigated the effects of C. asiatica water extract on activity of subtypes of phospholipase A(2) (PLA(2)) in primary cultures of rat cortical neurons and quantified by HPLC a possible molecule responsible for the activity. The cPLA(2) and sPLA(2) activities were inhibited in vitro by asiaticoside present in the water extract of C. asiatica. This extract may be a candidate for the treatment of neurodegenerative processes because of its pharmacological activity in the brain and its low toxicity, as attested by its long popular use as a natural product.

Ether-A -go-go 1 (Eag1) Potassium Channel Expression in Dopaminergic Neurons of Basal Ganglia is Modulated by 6-Hydroxydopamine Lesion

The ether A go-go (Eag) gene encodes the voltage-gated potassium (K+) ion channel Kv10.1, whose function still remains unknown. As dopamine may directly affect K+ channels, we evaluated whether a nigrostriatal dopaminergic lesion induced by the neurotoxin 6-hydroxydopamine (6-OHDA) would alter Eag1-K+ channel expression in the rat basal ganglia and related brain regions. Male Wistar rats received a microinjection of either saline or 6-OHDA (unilaterally) into the medial forebrain bundle. The extent of the dopaminergic lesion induced by 6-OHDA was evaluated by apomorphine-induced rotational behavior and by tyrosine hydroxylase (TH) immunoreactivity. The 6-OHDA microinjection caused a partial or complete lesion of dopaminergic cells, as well as a reduction of Eag1+ cells in a manner proportional to the extent of the lesion. In addition, we observed a decrease in TH immunoreactivity in the ipsilateral striatum. In conclusion, the expression of the Eag1-K+-channel throughout the nigrostriatal pathway in the rat brain, its co-localization with dopaminergic cells and its reduction mirroring the extent of the lesion highlight a physiological circuitry where the functional role of this channel can be investigated. The Eag1-K+ channel expression in dopaminergic cells suggests that these channels are part of the diversified group of ion channels that generate and maintain the electrophysiological activity pattern of dopaminergic midbrain neurons.

Differential effects of undernourishment on the differentiation and maturation of rat enteric neurons

The colocalization, number, and size of various classes of enteric neurons immunoreactive (IR) for the purinergic P2X2 and P2X7 receptors (P2X2R, P2X7R) were analyzed in the myenteric and submucosal plexuses of control, undernourished, and re-fed rats. Pregnant rats were exposed to undernourishment (protein-deprivation) or fed a control diet, and their offspring comprised the following experimental groups: rats exposed to a normal diet throughout gestation until postnatal day (P)42, rats protein-deprived throughout gestation and until P42, and rats protein-deprived throughout gestation until P21 and then given a normal diet until P42. Immunohistochemistry was performed on the myenteric and submucosal plexuses to evaluate immunoreactivity for P2X2R, P2X7R, nitric oxide synthase (NOS), choline acetyltransferase (ChAT), calbindin, and calretinin. Double-immunohistochemistry of the myenteric and submucosal plexuses demonstrated that 100% of NOS-IR, calbindin-IR, calretinin-IR, and ChAT-IR neurons in all groups also expressed P2X2R and P2X7R. Neuronal density increased in the myenteric and submucosal plexuses of undernourished rats compared with controls. The average size (profile area) of some types of neurons in the myenteric and submucosal plexuses was smaller in the undernourished than in the control animals. These changes appeared to be reversible, as animals initially undernourished but then fed a normal diet at P21 (re-feeding) were similar to controls. Thus, P2X2R and P2X7R are present in NOS-positive inhibitory neurons, calbindin- and calretinin-positive intrinsic primary afferent neurons, cholinergic secretomotor neurons, and vasomotor neurons in rats. Alterations in these neurons during undernourishment are reversible following re-feeding

Effects of ischemia and reperfusion on subpopulations of rat enteric neurons expressing the P2X7 receptor

BACKGROUND: Intestinal ischemia followed by reperfusion (I/R) may occur following intestinal obstruction. In rats, I/R in the small intestine leads to structural changes accompanied by neuronal death. AIM: To analyze the impact of I/R injury on different neuronal populations in the myenteric plexus of rat ileum. METHODS: The ileal artery was occluded for 35 min and animals were euthanized 6, 24, and 72 h, and 1 week later. Immunohistochemistry was performed with antibodies against the P2X7 receptor as well as nitric oxide synthase (NOS), calbindin, calretinin, choline acetyltransferase (ChAT), or the pan-neuronal marker anti-HuC/D. RESULTS: Double immunolabeling demonstrated that 100% of NOS-, calbindin-, calretinin-, and ChAT-immunoreactive neurons in all groups expressed the P2X7 receptor. Following I/R, neuronal density decreased by 22.6% in P2X7 receptor-immunoreactive neurons, and decreased by 46.7, 38, 39.8, 21.7, and 20% in NOS-, calbindin-, calretinin-, ChAT-, and HuC/D-immunoreactive neurons, respectively, at 6, 24, and 72 h and 1 week following injury compared to the control and sham groups. We also observed a 14% increase in the neuronal cell body profile area of the NOS-immunoreactive neurons at 6 and 24 h post-I/R and a 14% increase in ChAT-immunoreactive neurons at 1 week following I/R. However, the average size of the calretinin-immunoreactive neurons was reduced by 12% at 6 h post-I/R and increased by 8% at 24 h post-I/R. CONCLUSIONS: This work demonstrates that I/R is associated with a significant loss of different subpopulations of neurons in the myenteric plexus accompanied by morphological changes, all of which may underlie conditions related to intestinal motility disorder

Neurotoxicity of Anhydroecgonine Methyl Ester, a Crack Cocaine Pyrolysis Product

Smoking crack cocaine involves the inhalation of cocaine and its pyrolysis product, anhydroecgonine methyl ester (AEME). Although there is evidence that cocaine is neurotoxic, the neurotoxicity of AEME has never been evaluated. AEME seems to have cholinergic agonist properties in the cardiovascular system; however, there are no reports on its effects in the central nervous system. The aim of this study was to investigate the neurotoxicity of AEME and its possible cholinergic effects in rat primary hippocampal cell cultures that were exposed to different concentrations of AEME, cocaine, and a cocaineAEME combination. We also evaluated the involvement of muscarinic cholinergic receptors in the neuronal death induced by these treatments using concomitant incubation of the cells with atropine. Neuronal injury was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. The results of the viability assays showed that AEME is a neurotoxic agent that has greater neurotoxic potential than cocaine after 24 and 48 h of exposure. We also showed that incubation for 48 h with a combination of both compounds in equipotent concentrations had an additive neurotoxic effect. Although both substances decreased cell viability in the MTT assay, only cocaine increased LDH release. Caspase-3 activity was increased after 3 and 6 h of incubation with 1mM cocaine and after 6 h of 0.1 and 1.0mM AEME exposure. Atropine prevented the AEME-induced neurotoxicity, which suggests that muscarinic cholinergic receptors are involved in AEME's effects. In addition, binding experiments confirmed that AEME has an affinity for muscarinic cholinergic receptors. Nevertheless, atropine was not able to prevent the neurotoxicity produced by cocaine and the cocaineAEME combination, suggesting that these treatments activated other neuronal death pathways. Our results suggest a higher risk for neurotoxicity after smoking crack cocaine than after cocaine use alone.

Stimulation of peripheral Kappa opioid receptors inhibits inflammatory hyperalgesia via activation of the PI3K gamma/AKT/nNOS/NO signaling pathway

Background: In addition to their central effects, opioids cause peripheral analgesia. There is evidence showing that peripheral activation of kappa opioid receptors (KORs) inhibits inflammatory pain. Moreover, peripheral mu-opioid receptor (MOR) activation are able to direct block PGE(2)-induced ongoing hyperalgesia However, this effect was not tested for KOR selective activation. In the present study, the effect of the peripheral activation of KORs on PGE(2)-induced ongoing hyperalgesia was investigated. The mechanisms involved were also evaluated. Results: Local (paw) administration of U50488 (a selective KOR agonist) directly blocked, PGE(2)-induced mechanical hyperalgesia in both rats and mice. This effect was reversed by treating animals with L-NMMA or N-propyl-L-arginine (a selective inhibitor of neuronal nitric oxide synthase, nNOS), suggesting involvement of the nNOS/NO pathway. U50488 peripheral effect was also dependent on stimulation of PI3K gamma/AKT because inhibitors of these kinases also reduced peripheral antinociception induced by U50488. Furthermore, U50488 lost its peripheral analgesic effect in PI3K gamma null mice. Observations made in vivo were confirmed after incubation of dorsal root ganglion cultured neurons with U50488 produced an increase in the activation of AKT as evaluated by western blot analyses of its phosphorylated form. Finally, immunofluorescence of DRG neurons revealed that KOR-expressing neurons also express PI3K gamma (congruent to 43%). Conclusions: The present study indicates that activation of peripheral KORs directly blocks inflammatory hyperalgesia through stimulation of the nNOS/NO signaling pathway which is probably stimulated by PI3K gamma/AKT signaling. This study extends a previously study of our group suggesting that PI3K gamma/AKT/nNOS/NO is an important analgesic pathway in primary nociceptive neurons.

Opioidergic, GABAergic and serotonergic neurotransmission in the dorsal raphe nucleus modulates tonic immobility in guinea pigs

Tonic immobility (TI) is an innate defensive behavior that can be elicited by physical restriction and postural inversion and is characterized by a profound and temporary state of akinesis. Our previous studies demonstrated that the stimulation of serotonin receptors in the dorsal raphe nucleus (DRN) appears to be biphasic during TI responses in guinea pigs (Cavia porcellus). Serotonin released by the DRN modulates behavioral responses and its release can occur through the action of different neurotransmitter systems, including the opioidergic and GABAergic systems. This study examines the role of opioidergic, GABAergic and serotonergic signaling in the DRN in TI defensive behavioral responses in guinea pigs. Microinjection of morphine (1.1 nmol) or bicuculline (0.5 nmol) into the DRN increased the duration of TI. The effect of morphine (1.1 nmol) was antagonized by pretreatment with naloxone (0.7 nmol), suggesting that the activation of pi opioid receptors in the DRN facilitates the TI response. By contrast, microinjection of muscimol (0.5 nmol) into the DRN decreased the duration of TI. However, a dose of muscimol (0.26 nmol) that alone did not affect TI, was sufficient to inhibit the effect of morphine (1.1 nmol) on TI, indicating that GABAergic and enkephalinergic neurons interact in the DRN. Microinjection of alpha-methyl-5-HT (1.6 nmol), a 5-HT2 agonist, into the DRN also increased TI. This effect was inhibited by the prior administration of naloxone (0.7 nmol). Microinjection of 8-OH-DPAT (1.3 nmol) also blocked the increase of TI promoted by morphine (1.1 nmol). Our results indicate that the opioidergic, GABAergic and serotonergic systems in the DRN are important for modulation of defensive behavioral responses of TI. Therefore, we suggest that opioid inhibition of GABAergic neurons results in disinhibition of serotonergic neurons and this is the mechanism by which opioids could enhance TI. Conversely, a decrease in TI could occur through the activation of GABAergic interneurons. (C) 2012 Elsevier Inc. All rights reserved.

Phenotypic alterations of neuropeptide Y and calcitonin gene-related peptide-containing neurons innervating the rat temporomandibular joint during carrageenan-induced arthritis

The aim of this study was to identify immunoreactive neuropeptide Y (NPY) and calcitonin gene-related peptide (CGRP) neurons in the autonomic and sensory ganglia, specifically neurons that innervate the rat temporomandibular joint (TMJ). A possible variation between the percentages of these neurons in acute and chronic phases of carrageenan-induced arthritis was examined. Retrograde neuronal tracing was combined with indirect immunofluorescence to identify NPY-immuno-reactive (NPY-IR) and CGRP-immunoreactive (CGRP-IR) neurons that send nerve fibers to the normal and arthritic temporomandibular joint. In normal joints, NPY-IR neurons constitute 78 +/- 3%, 77 +/- 6% and 10 +/- 4% of double-labeled nucleated neuronal profile originated from the superior cervical, stellate and otic ganglia, respectively. These percentages in the sympathetic ganglia were significantly decreased in acute (58 +/- 2% for superior cervical ganglion and 58 +/- 8% for stellate ganglion) and chronic (60 +/- 2% for superior cervical ganglion and 59 +/- 15% for stellate ganglion) phases of arthritis, while in the otic ganglion these percentages were significantly increased to 19 +/- 5% and 13 +/- 3%, respectively. In the trigeminal ganglion, CGRP-IR neurons innervating the joint significantly increased from 31 +/- 3% in normal animals to 54 +/- 2% and 49 +/- 3% in the acute and chronic phases of arthritis, respectively. It can be concluded that NPY neurons that send nerve fibers to the rat temporomandibular joint are located mainly in the superior cervical, stellate and otic ganglia. Acute and chronic phases of carrageenan-induced arthritis lead to an increase in the percentage of NPY-IR parasympathetic and CGRP-IR sensory neurons and to a decrease in the percentage of NPY-IR sympathetic neurons related to TMJ innervation.

Role of transient receptor potential vanilloid 4 in rat joint inflammation

Objective To determine whether activation of transient receptor potential vanilloid 4 (TRPV-4) induces inflammation in the rat temporomandibular joint (TMJ), and to assess the effects of TRPV-4 agonists and proinflammatory mediators, such as a protease-activated receptor 2 (PAR-2) agonist, on TRPV-4 responses. Methods Four hours after intraarticular injection of carrageenan into the rat joints, expression of TRPV-4 and PAR-2 in trigeminal ganglion (TG) neurons and in the TMJs were evaluated by real-time reverse transcriptionpolymerase chain reaction and immunofluorescence, followed by confocal microscopy. The functionality of TRPV-4 and its sensitization by a PAR-2activating peptide (PAR-2AP) were analyzed by measuring the intracellular Ca2+ concentration in TMJ fibroblast-like synovial cells or TG neurons. Plasma extravasation, myeloperoxidase activity, and the head-withdrawal threshold (index of mechanical allodynia) were evaluated after intraarticular injection of selective TRPV-4 agonists, either injected alone or coinjected with PAR-2AP. Results In the rat TMJs, TRPV-4 and PAR-2 expression levels were up-regulated after the induction of inflammation. Two TRPV-4 agonists specifically activated calcium influx in TMJ fibroblast-like synovial cells or TG neurons. In vivo, the agonists triggered dose-dependent increases in plasma extravasation, myeloperoxidase activity, and mechanical allodynia. In synovial cells or TG neurons, pretreatment with PAR-2AP potentiated a TRPV-4 agonistinduced increase in [Ca2+]i. In addition, TRPV-4 agonistinduced inflammation was potentiated by PAR-2AP in vivo. Conclusion In this rat model, TRPV-4 is expressed and functional in TG neurons and synovial cells, and activation of TRPV-4 in vivo causes inflammation in the TMJ. Proinflammatory mediators, such as PAR-2 agonists, sensitize the activity of TRPV-4. These results identify TRPV-4 as an important signal of inflammation in the joint.

PARAVENTRICULAR AND SUPRAOPTIC NUCLEI OF THE HYPOTHALAMUS MEDIATE CARDIOVASCULAR RESPONSES EVOKED BY THE MICROINJECTION OF NORADRENALINE INTO THE MEDIAL AMYGDALOID NUCLEUS OF THE RAT BRAIN

The medial amygdaloid nucleus (MeA) is a part of the limbic system and is involved in cardiovascular modulation. We previously reported that microinjection of noradrenaline (NA) into the MeA of unanesthetized rats caused pressor and bradycardiac responses, which were mediated by acute vasopressin release into the systemic circulation. In the present study, we tested the possible involvement of magnocellular neurons of the paraventricular (PVN) and/or supraoptic (SON) of the hypothalamus that synthesize vasopressin in the cardiovascular pathway activated by the microinjection of NA into the MeA. Pressor and bradycardiac responses to the microinjection of NA (27 nmol/100 nL) into the MeA were blocked by pretreatment of either the PVN or the SON with cobalt chloride (CoCl2, 1 mM/100 nL), thus indicating that both hypothalamic nuclei mediate the cardiovascular responses evoked by microinjection of NA Into the MeA. Our results suggest that the pressor and bradycardiac response caused by the microinjection of NA into the MeA is mediated by magnocellular neurons in both the PVN and SON. (C) 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

Differential projections from the lateral habenula to the rostromedial tegmental nucleus and ventral tegmental area in the rat

The mesopontine rostromedial tegmental nucleus (RMTg) is a mostly ?-aminobutyric acid (GABA)ergic structure believed to be a node for signaling aversive events to dopamine (DA) neurons in the ventral tegmental area (VTA). The RMTg receives glutamatergic inputs from the lateral habenula (LHb) and sends substantial GABAergic projections to the VTA, which also receives direct projections from the LHb. To further specify the topography of LHb projections to the RMTg and VTA, small focal injections of the anterograde tracer Phaseolus vulgaris leucoagglutinin were aimed at different subdivisions of the LHb. The subnuclear origin of LHb inputs to the VTA and RMTg was then confirmed by injections of the retrograde tracer cholera toxin subunit b into the VTA or RMTg. Furthermore, we compared the topographic position of retrogradely labeled neurons in the RMTg resulting from VTA injections with that of anterogradely labeled axons emerging from the LHb. As revealed by anterograde and retrograde tracing, LHb projections were organized in a strikingly topographic manner, with inputs to the RMTg mostly arising from the lateral division of the LHb (LHbL), whereas inputs to the VTA mainly emerged from the medial division of the LHb (LHbM). In the RMTg, profusely branched LHb axons were found in close register with VTA projecting neurons and were frequently apposed to the latter. Overall, our findings demonstrate that LHb inputs to the RMTg and VTA arise from different divisions of the LHb and provide direct evidence for a disynaptic pathway that links the LHbL to the VTA via the RMTg. J. Comp. Neurol. 520:12781300, 2012. (C) 2011 Wiley Periodicals, Inc.

Amitriptyline prolongs the antihyperalgesic effect of 2- or 100-Hz electro-acupuncture in a rat model of post-incision pain

The mechanisms through which electro-acupuncture (EA) and tricyclic antidepressants produce analgesia seem to be complementary: EA inhibits the transmission of noxious messages by activating supraspinal serotonergic and noradrenergic neurons that project to the spinal cord, whereas tricyclic antidepressants affect pain transmission by inhibiting the reuptake of norepinephrine and serotonin at the spinal level. This study utilized the tail-flick test and a model of post-incision pain to compare the antihyperalgesic effects of EA at frequencies of 2 or 100 Hz in rats treated with intraperitoneal or intrathecal amitriptyline (a tricyclic antidepressant). A gradual increase in the tail-flick latency (TFL) occurred during a 20-min period of EA. A strong and long-lasting reduction in post-incision hyperalgesia was observed after stimulation; the effect after 2 Hz lasting longer than after 100-Hz EA. Intraperitoneal or intrathecal amitriptyline potentiated the increase in TFL in the early moments of 2- or 100-Hz EA, and the intensity of the antihyperalgesic effect of 100-Hz EA in both the incised and non-incised paw. In contrast, it did not significantly change the intensity of the antihyperalgesic effect of 2-Hz EA. The EA-induced antihyperalgesic effects lasted longer after intraperitoneal or intrathecal amitriptyline than after saline, with this effect of amitriptyline being more evident after 100-than after 2-Hz EA. The synergetic effect of amitriptyline and EA against post-incision pain shown here may therefore represent an alternative for prolonging the efficacy of EA in the management of post-surgical clinical pain.

Neural differentiation of rat aorta pericyte cells

Pericyte perivascular cells, believed to originate mesenchymal stem cells (MSC), are characterized by their capability to differentiate into various phenotypes and participate in tissue reconstruction of different organs, including the brain. We show that these cells can be induced to differentiation into neural-like phenotypes. For these studies, pericytes were obtained from aorta ex-plants of Sprague-Dawley rats and differentiated into neural cells following induction with trans retinoic acid (RA) in serum-free defined media or differentiation media containing nerve growth and brain-derived neuronal factor, B27, N2, and IBMX. When induced to differentiation with RA, cells express the pluripotency marker protein stage-specific embryonic antigen-1, neural-specific proteins beta 3-tubulin, neurofilament-200, and glial fibrillary acidic protein, suggesting that pericytes undergo differentiation, similar to that of neuroectodermal cells. Differentiated cells respond with intracellular calcium transients to membrane depolarization by KCl indicating the presence of voltage-gated ion channels and express functional N-methyl-D-aspartate receptors, characteristic for functional neurons. The study of neural differentiation of pericytes contributes to the understanding of induction of neuroectodermal differentiation as well as providing a new possible stem-cell source for cell regeneration therapy in the brain. (C) 2011 International Society for Advancement of Cytometry

Chronic infusion of amyloid-β peptide and sustained attention altered α7 nicotinic receptor density in the rat brain

It is already known that progressive degeneration of cholinergic neurons in brain areas such as the hippocampus and the cortex leads to memory deficits, as observed in Alzheimer's disease. This work verified the effects of the infusion of amyloid-beta (A beta) peptide associated to an attentional rehearsal on the density of alpha 7 nicotinic cholinergic receptor (nAChR) in the brain of male Wistar rats. Animals received intracerebroventricular infusion of A beta or vehicle (control - C) and their attention was stimulated weekly (Stimulated A beta group: S-A beta and Stimulated Control group: SC) or not (Non-Stimulated A beta group: N-SA beta and Non-Stimulated Control group: N-SC), using an active avoidance apparatus. Conditioned avoidance responses (CAR) were registered. Chronic infusion of A beta caused a 37% reduction in CAR for N-SA beta. In S-A beta, this reduction was not observed. At the end, brains were extracted and autoradiography for alpha 7 nAChR was conducted using [I-125]-alpha-bungarotoxin. There was an increase in alpha 7 density in hippocampus, cortex and amygdala of SA beta animals, together with the memory preservation. In recent findings from our lab using mice infused with A beta and the alpha 7 antagonist methyllycaconitine, and stimulated weekly in the same apparatus, it was observed that memory maintenance was abolished. So, the increase in alpha 7 density in brain areas related to memory might be related to a participation of this receptor in the long-lasting change in synaptic plasticity, which is important to improve and maintain memory consolidation.

Effect of caloric restriction on myenteric neuroplasticity in the rat duodenum during aging

The objective of this study was to evaluate the effects of caloric restriction (CR) on myenteric neurons in the duodenum of Wistar rats during aging. Thirty rats were divided into three groups: the C group (six-month-old animals that were fed a normal diet from weaning until six months of age), the SR group (18-month-old animals that were fed a normal diet from weaning until 18 months of age) and the CR group (18-month-old animals that were fed a 30% CR diet after six months of age). After 12 months, the animals were euthanized. Whole-mount preparations of the duodenums were either stained with Giemsa or underwent NADPH-diaphorase histochemistry to determine the general myenteric neuron population and the nitrergic neuron subpopulation (NADPH-d +), respectively. The NADPH-d-negative (NADPH-d -) neuron population was estimated based on the difference between the Giemsa-stained and NADPH-d + neurons. The neurons were counted, and the cell body areas were measured. Aging was associated with neuronal loss in the SR group, which was minimized by caloric restriction in the CR group. The density (mm(2)) of the Giemsa-stained neurons was higher in the SR group (79.09 +/- 6.25) than in the CR (92.37 +/- 11.6) and C (111.68 +/- 15.26) groups. The density of the NADPH-d + neurons was higher in the SR group (44.90 +/- 5.88) than in the C (35.75 +/- 1.6) and RC (39.14 +/- 7.02) groups. The density of NADPH-d - neurons was higher in the CR (49.73 +/- 12.08) and C (75.64 +/- 17.05) groups than in the SR group (33.82 +/- 4.5). In the C group, 32% and 68% of the Giemsa-stained myenteric neurons were NADPH-d + or NADPH-d -, respectively. With aging (SR group), the percentage of nitrergic neurons (56.77%) increased, whereas the percentage of NADPH-d - neurons (43.22%) decreased. In the CR group, the change in the percentage of nitrergic (42.37%) and NADPH-d - (57.62%) neurons was lower. As NADPH-d - neurons will be mostly cholinergic neurons, CR appears to reduce the loss of cholinergic neurons during aging. The cell body dimensions (mu m(2)) were not altered by aging or CR. Thus. CR had a protective effect on myenteric neurons during aging. (C) 2012 Elsevier B.V. All rights reserved.