Jonathon L. McClain

Ca2+ Responses in Enteric Glia Are Mediated by Connexin-43 Hemichannels and Modulate Colonic Transit in Mice

BACKGROUND & AIMS In the enteric nervous system, neurotransmitters initiate changes in calcium (Ca(2+) responses) in glia, but it is not clear how this process affects intestinal function. We investigated whether Ca(2+)-mediated responses in enteric glia are required to maintain gastrointestinal function. METHODS We used in situ Ca(2+) imaging to monitor glial Ca(2+) responses, which were manipulated with pharmacologic agents or via glia-specific disruption of the gene encoding connexin-43 (Cx43) (hGFAP::CreER(T2+/-)/Cx43(f/f) mice). Gastrointestinal function was assessed based on pellet output, total gut transit, colonic bead expulsion, and muscle tension recordings. Proteins were localized and quantified by immunohistochemistry, immunoblot, and reverse transcription polymerase chain reaction analyses. RESULTS Ca(2+) responses in enteric glia of mice were mediated by Cx43 hemichannels. Cx43 immunoreactivity was confined to enteric glia within the myenteric plexus of the mouse colon; the Cx43 inhibitors carbenoxolone and 43Gap26 inhibited the ability of enteric glia to propagate Ca(2+) responses. In vivo attenuation of Ca(2+) responses in the enteric glial network slowed gut transit overall and delayed colonic transit--these changes are also observed during normal aging. Altered motility with increasing age was associated with reduced glial Ca(2+)-mediated responses and changes in glial expression of Cx43 messenger RNA and protein. CONCLUSIONS Ca(2+)-mediated responses in enteric glia regulate gastrointestinal function in mice. Altered intercellular signaling between enteric glia and neurons might contribute to motility disorders.

Doxycycline, a matrix metalloprotease inhibitor, reduces vascular remodeling and damage after cerebral ischemia in stroke-prone spontaneously hypertensive rats.

Matrix metalloproteases (MMPs) are a family of zinc peptidases involved in extracellular matrix turnover. There is evidence that increased MMP activity is involved in remodeling of resistance vessels in chronic hypertension. Thus we hypothesized that inhibition of MMP activity with doxycycline (DOX) would attenuate vascular remodeling. Six-week-old male stroke-prone spontaneously hypertensive rats (SHRSP) were treated with DOX (50 mg·kg(-1)·day(-1) in the drinking water) for 6 wk. Untreated SHRSP were controls. Blood pressure was measured by telemetry during the last week. Middle cerebral artery (MCA) and mesenteric resistance artery (MRA) passive structures were assessed by pressure myography. MMP-2 expression in aortas was measured by Western blot. All results are means ± SE. DOX caused a small increase in mean arterial pressure (SHRSP, 154 ± 1; SHRSP + DOX, 159 ± 3 mmHg; P < 0.001). Active MMP-2 expression was reduced in aorta from SHRSP + DOX (0.21 ± 0.06 vs. 0.49 ± 0.13 arbitrary units; P < 0.05). In the MCA, at 80 mmHg, DOX treatment increased the lumen (273.2 ± 4.7 vs. 238.3 ± 6.3 μm; P < 0.05) and the outer diameter (321 ± 5.3 vs. 290 ± 7.6 μm; P < 0.05) and reduced the wall-to-lumen ratio (0.09 ± 0.002 vs. 0.11 ± 0.003; P < 0.05). Damage after transient cerebral ischemia (transient MCA occlusion) was reduced in SHRSP + DOX (20.7 ± 4 vs. 45.5 ± 5% of hemisphere infarcted; P < 0.05). In the MRA, at 90 mmHg DOX, reduced wall thickness (29 ± 1 vs. 22 ± 1 μm; P < 0.001) and wall-to-lumen ratio (0.08 ± 0.004 vs. 0.11 ± 0.008; P < 0.05) without changing lumen diameter. These results suggest that MMPs are involved in hypertensive vascular remodeling in both the peripheral and cerebral vasculature and that DOX reduced brain damage after cerebral ischemia.

Enteric glia mediate neuron death in colitis through purinergic pathways that require connexin-43 and nitric oxide.

Background & Aims The concept of enteric glia as regulators of intestinal homeostasis is slowly gaining acceptance as a central concept in neurogastroenterology. Yet how glia contribute to intestinal disease is still poorly understood. Purines generated during inflammation drive enteric neuron death by activating neuronal P2X7 purine receptors (P2X7R); triggering adenosine triphosphate (ATP) release via neuronal pannexin-1 channels that subsequently recruits intracellular calcium ([Ca2+]i) in surrounding enteric glia. We tested the hypothesis that the activation of enteric glia contributes to neuron death during inflammation. Methods We studied neuroinflammation in vivo using the 2,4-dinitrobenzene sulfonic acid model of colitis and in situ using whole-mount preparations of human and mouse intestine. Transgenic mice with a targeted deletion of glial connexin-43 (Cx43) [GFAP::CreERT2+/−/Cx43f/f] were used to specifically disrupt glial signaling pathways. Mice deficient in inducible nitric oxide (NO) synthase (iNOS−/−) were used to study NO production. Protein expression and oxidative stress were measured using immunohistochemistry and in situ Ca2+ and NO imaging were used to monitor glial [Ca2+]i and [NO]i. Results Purinergic activation of enteric glia drove [Ca2+]i responses and enteric neuron death through a Cx43-dependent mechanism. Neurotoxic Cx43 activity, driven by NO production from glial iNOS, was required for neuron death. Glial Cx43 opening liberated ATP and Cx43-dependent ATP release was potentiated by NO. Conclusions Our results show that the activation of glial cells in the context of neuroinflammation kills enteric neurons. Mediators of inflammation that include ATP and NO activate neurotoxic pathways that converge on glial Cx43 hemichannels. The glial response to inflammatory mediators might contribute to the development of motility disorders.

Agonist-Evoked Ca2+ Signaling in Enteric Glia Drives Neural Programs That Regulate Intestinal Motility in Mice

Background & Aims Gastrointestinal motility is regulated by enteric neural circuitry that includes enteric neurons and glia. Enteric glia monitor synaptic activity and exhibit responses to neurotransmitters that are encoded by intracellular calcium (Ca2+) signaling. What role evoked glial responses play in the neural regulation of gut motility is unknown. We tested how evoking Ca2+ signaling in enteric glia affects the neural control of intestinal motility. Methods We used a novel chemogenetic mouse model that expresses the designer receptor hM3Dq under the transcriptional control of the glial fibrillary acidic protein (GFAP) promoter (GFAP::hM3Dq mice) to selectively trigger glial Ca2+ signaling. We used in situ Ca2+ imaging and immunohistochemistry to validate this model, and we assessed gut motility by measuring pellet output and composition, colonic bead expulsion time, small intestinal transit time, total gut transit time, colonic migrating motor complex (CMMC) recordings, and muscle tension recordings. Results Expression of the hM3Dq receptor is confined to GFAP-positive enteric glia in the intestines of GFAP::hM3Dq mice. In these mice, application of the hM3Dq agonist clozapine-N-oxide (CNO) selectively triggers intracellular Ca2+ responses in enteric glia. Glial activation drove neurogenic contractions in the ileum and colon but had no effect on neurogenic relaxations. CNO enhanced the amplitude and frequency of CMMCs in ex vivo preparations of the colon, and CNO increased colonic motility in vivo. CNO had no effect on the composition of fecal matter, small intestinal transit, or whole gut transit. Conclusions Glial excitability encoded by intracellular Ca2+ signaling functions to modulate excitatory enteric circuits. Selectively triggering glial Ca2+ signaling might be a novel strategy to improve gut function in motility disorders.

Tempol, a superoxide dismutase mimetic, prevents cerebral vessel remodeling in hypertensive rats

Increased reactive oxygen species (ROS) production is involved in the pathogenesis of hypertension and stroke. The effects of ROS on cerebral vessels from hypertensive rats have not been studied. We hypothesized that tempol, a superoxide dismutase mimetic, would prevent middle cerebral artery (MCA) remodeling in stroke-prone spontaneously hypertensive rats (SHRSP). Six-week-old male SHRSP were treated with tempol (1mM) for 6weeks. The MCA was then removed and mounted in a pressure myograph to study tone generation, vessel reactivity, and passive vessel structure. Data are shown as mean±SEM, tempol vs. control. Plasma thiobarbituric acid reactive substances (TBARS) were decreased by tempol treatment (14.15±1.46 vs. 20.55±1.25nM of malondialdehyde [MDA]/ml, p=0.008). Maximum serotonin-induced constriction was increased by tempol treatment, without changes in dilation to adenosine diphosphate or tone generation. At an intralumenal pressure of 80mmHg, tempol caused a dramatic increase in the MCA lumen diameter (246±5 vs. 207±3μm, p<0.001), outer diameter (281±5 vs. 241±3μm, p<0.001), lumen cross-sectional area, and vessel cross-sectional area. Collagen IV mRNA expressions were increased by 2.4-fold after tempol treatment. These results suggest that ROS are involved in the remodeling of the cerebral vasculature of SHRSP and that ROS scavenging can attenuate this process.

Improvement in middle cerebral artery structure and endothelial function in stroke-prone spontaneously hypertensive rats after macrophage depletion

Inflammation is involved in the pathogenesis of hypertension. Hypertensive animals have an increased number of perivascular macrophages in cerebral arteries. Macrophages might be involved in remodeling of the cerebral vasculature. We hypothesized that peripheral macrophage depletion would improve MCA structure and function in hypertensive rats.

The Development of Hypertension and Hyperaldosteronism in a Rodent Model of Life-Long Obesity

Aldosterone has been linked to the deleterious cardiovascular effects of obesity in humans. The association of aldosterone with obesity in rodents is less well defined, particularly in models of diet-induced obesity. We hypothesized that adrenal aldosterone production and aldosterone synthase expression would be increased in rats with obesity-induced hypertension. Male Sprague Dawley rats were fed a high-fat (HF: 36% fat) or control diet from 3 wk of age, and mean arterial pressure (MAP) was measured by telemetry. MAP was increased after 4 wk of HF diet; this was 6 wk before changes in body weight. Mineralocorticoid receptor antagonism did not prevent the HF-induced increase in MAP. After 17 wk on the diets, HF rats had increased body and fat weights (abdominal and epididymal) and were insulin resistant (Homeostasis Model Assessment index: 3.53 ± 0.43 vs. 8.52 ± 1.77; control vs. HF, P < 0.05). Plasma aldosterone levels were increased in the HF rats (64.14 ± 14.96 vs. 206.25 ± 47.55 pg/ml; control vs. HF, P < 0.05). This occurred independently of plasma renin activity (4.8 ± 0.92 vs. 4.73 ± 0.66 ng/ml/h, control vs. HF). The increase in aldosterone was accompanied by a 2-fold increase in adrenal aldosterone synthase mRNA expression and zona glomerulosa hypertrophy. Rats were also studied after 8 wk of HF diet, a time when MAP, but not body weight, was increased. At this time plasma aldosterone was unchanged but plasma renin activity was increased (4.4 ± 0.5 vs. 8.1 ± 1.3 ng/ml/h; control vs. HF, P < 0.05). These studies suggest that rats fed a HF diet from weaning may be a useful model for studying obesity-associated hyperaldosteronism.

Tumor necrosis factor-α inhibition attenuates middle cerebral artery remodeling but increases cerebral ischemic damage in hypertensive rats

Hypertension causes vascular inflammation evidenced by an increase in perivascular macrophages and proinflammatory cytokines in the arterial wall. Perivascular macrophage depletion reduced tumor necrosis factor (TNF)-α expression in cerebral arteries of hypertensive rats and attenuated inward remodeling, suggesting that TNF-α might play a role in the remodeling process. We hypothesized that TNF-α inhibition would improve middle cerebral artery (MCA) structure and reduce damage after cerebral ischemia in hypertensive rats. Six-week-old male stroke-prone spontaneously hypertensive rats (SHRSP) were treated with the TNF-α inhibitor etanercept (ETN; 1.25 mg·kg(-1)·day(-1) ip daily) or PBS (equivolume) for 6 wk. The myogenic tone generation, postischemic dilation, and passive structure of MCAs were assessed by pressure myography. Cerebral ischemia was induced by MCA occlusion (MCAO). Myogenic tone was unchanged, but MCAs from SHRSP + ETN had larger passive lumen diameter and reduced wall thickness and wall-to-lumen ratio. Cerebral infarct size was increased in SHRSP + ETN after transient MCAO, despite an improvement in dilation of nonischemic MCA. The increase in infarct size was linked to a reduction in the number of microglia in the infarct core and upregulation of markers of classical macrophage/microglia polarization. There was no difference in infarct size after permanent MCAO or when untreated SHRSP subjected to transient MCAO were given ETN at reperfusion. Our data suggests that TNF-α inhibition attenuates hypertensive MCA remodeling but exacerbates cerebral damage following ischemia/reperfusion injury likely due to inhibition of the innate immune response of the brain.

The acute inhibition of enteric glial metabolism with fluoroacetate alters calcium signaling, hemichannel function and the expression of key proteins.

Glia play key roles in the regulation of neurotransmission in the nervous system. Fluoroacetate (FA) is a metabolic poison widely used to study glial functions by disrupting the tricarboxylic acid cycle enzyme aconitase. Despite the widespread use of FA, the effects of FA on essential glial functions such as calcium (Ca2+) signaling and hemichannel function remain unknown. Therefore, our goal was to assess specifically the impact of FA on essential glial cell functions that are involved with neurotransmission in the enteric nervous system. To this end, we generated a new optogenetic mouse model to study specifically the effects of FA on enteric glial Ca2+ signaling by crossing PC::G5-tdTomato mice with Sox10::creERT2 mice. FA did not change the peak glial Ca2+ response when averaged across all glia within a ganglion. However, FA decreased the percent of responding glia by 30% (P < 0.05) and increased the peak Ca2+ response of the glial cells that still exhibited a response by 26% (P < 0.01). Disruption of Ca2+ signaling with FA impaired the activity-dependent uptake of ethidium bromide through connexin-43 (Cx43) hemichannels (P < 0.05) but did not affect baseline Cx43-dependent dye uptake. FA did not cause overt glial or neurodegeneration, but glial cells significantly increased glial fibrillary acid protein by 56% (P < 0.05) following treatment with FA. Together, these data show that the acute impairment of glial metabolism with FA causes key changes in glial functions associated with their roles in neurotransmission and phenotypic changes indicative of reactive gliosis. NEW & NOTEWORTHY Our study shows that the acute impairment of enteric glial metabolism with fluoroacetate (FA) alters specific glial functions that are associated with the modification of neurotransmission in the gut. These include subtle changes to glial agonist-evoked calcium signaling, the subsequent disruption of connexin-43 hemichannels, and changes in protein expression that are consistent with a transition to reactive glia. These changes in glial function offer a mechanistic explanation for the effects of FA on peripheral neuronal networks.

Temporary mineralocorticoid receptor antagonism during the development of hypertension improves cerebral artery dilation

Hypertension causes cerebral artery remodeling and increases the risk of stroke. Renin angiotensin system blockade during the development of hypertension has therapeutic effects even after treatment withdrawal. Mineralocorticoid receptor (MR) activation has been implicated in artery remodeling and impaired endothelial function. The possibility that there is a critical therapeutic window for MR antagonism has not been investigated. We hypothesized that temporary MR antagonism while hypertension develops would improve middle cerebral artery (MCA) structure and function in stroke-prone spontaneously hypertensive rats (SHRSP), even after treatment withdrawal. Six-week-old SHRSP were treated with spironolactone (25 mg/kg/day) from 6 to 12 weeks and when aged to 18 weeks, these rats were compared to age-matched untreated SHRSP. Surprisingly, temporary spironolactone treatment reduced the MCA outer and lumen diameter but had no effect on the wall thickness. Temporary spironolactone treatment improved nitric oxide and endothelium-derived hyperpolarizing factor mediated dilation but had no effect on blood pressure. Spironolactone treatment caused a very small reduction in the damage caused by permanent focal cerebral ischemia. These results suggest that temporary MR antagonism during the development of hypertension has divergent effects on the MCA, in that it causes a potentially detrimental reduction in the lumen diameter while improving vasodilation.