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Neuroscience Letters 587 (2015) 22–28

Contents lists available at ScienceDirect

Neuroscience Letters

jo ur nal ho me p age: www.elsev ier .com/ locate /neule t

esearch article

icroglial P2X7 receptor in the hypothalamic paraventricular nucleiontributes to sympathoexcitatory responses in acute myocardialnfarction rat

ongshu Du a,1, Meiyan Jiang c,1, Min Liu b,1, Jin Wang b, Chunmei Xia b, Ruijuan Guan b,inlin Shen b, Yonghua Ji a, Danian Zhu b,∗

Laboratory of Neuropharmacology and Neurotoxicology, Shanghai University, Shanghai 200444, PR ChinaDepartment of Physiology and Pathophysiology, Shanghai Medical College of Fudan University, 138 Yixueyuan Road, Shanghai 200032, PR ChinaOregon Hearing Research Center, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA

i g h l i g h t s

Microglial P2X7R in the PVN can be activated by endogenous ATP in rats with AMI.Activated P2X7R might sensitise oxytocinergic (OT) and vasopressin (AVP) neurons.Sensitised OT and AVP neurons enhanced the peripheric renal sympathetic nerve tone.

r t i c l e i n f o

rticle history:eceived 21 November 2013eceived in revised form 3 December 2014ccepted 12 December 2014vailable online 15 December 2014

eywords:ypothalamic paraventricular nucleusicroglia

2X7 receptorroinflammatory cytokinesxytocinergic and vasopressinergic neuron

a b s t r a c t

Several pieces of evidence indicate that the microglial P2X7 receptor (P2X7R) regulate cardiovascularactivities. We explored the possible roles of microglial P2X7R in the PVN mediated sympathoexcitatoryresponses in acute myocardial infarction (AMI) rat. Sprague–Dawley rats underwent coronary arteryligation to induce AMI. The rats received intraperitoneal administration of the P2X7R antagonist BrilliantBlue-G (BBG, 25 or 50 mg kg−1, once a day for 5 days) prior to myocardial ischemia. Other rats receivedbilateral microinjection of P2X7R-siRNA (0.015 or 0.03 nmol 0.1 �l per side, once a day for 2 days) targetingP2X7R mRNA into the PVN prior to myocardial ischemia. First, we examined the ATP levels and proteinexpression P2X7R in the PVN in different ischemia time groups, and we found that the change of P2X7R waspositive correlated with the ATP levels in a time-dependent manner. The double-immunofluorescenceevidence showed that P2X7R was mainly co-localizated with the microglial marker Iba-1 in the PVN.Second, gene knockdown of P2X7R with P2X7-siRNA or inhibition of P2X7R with BBG reduce the mRNA

cute myocardial infarction and protein expression of IL-1� and TNF-� in the PVN of AMI rat. Third, microinjected P2X7-siRNA alsosuppressed the up-regulation of P2X7R, oxytocin and vasopressin in the PVN of AMI rats. Fourth, P2X7-siRNA and BBG also attenuated the renal sympathetic nerve activity (RSNA) in the AMI rats. Our resultsindicate that microglial P2X7R activation in PVN mediating the production of proinflammatory cytokinesthat activate oxytocinergic and vasopressinergic neuron, which augmented the RSNA in the AMI rat.

© 2014 Elsevier Ireland Ltd. All rights reserved.

. Introduction

There is increasing assessment of the effects of proinflam-atory cytokines on cardiovascular and cardiovascular-related

njury, including acute myocardial infarction (AMI) and hyperten-

∗ Corresponding author. Tel.: +86 21 54237405; fax: +86 21 54237405.E-mail address: sdhzdds@126.com (D. Zhu).

1 Equal contributors.

ttp://dx.doi.org/10.1016/j.neulet.2014.12.026304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.

sion [9,16,21]. Especially, recent studies have provided evidenceto support a causal relationship between inflammation in thehypothalamic paraventricular nucleus (PVN) and the activation ofthe sympathetic nervous system [4,5]. However, how do the inflam-matory mediators form in the PVN and how do the inflammatorymediators enhance sympathetic nervous system activity in AMI ratremain unknown. In this study, we investigated a putative mecha-

nism.

Interleukin 1� (IL-1�) and tumor necrosis factor-� (TNF-�),well-recognized markers of inflammation, are too large to cross

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he blood-brain barrier. However, is there one feasible pathwayediated proinflammatory cytokines formation in PVN?

Microglia is a type of glial cell, which plays a critical role as aesident immunocompetent cell during ischemia [15,17]. Extra-ellular ATP triggers microglial activation through P2 receptorsn a animal disease model [13]. Activated microglia release sub-tances such as oxygen radicals [20], proinflammatory cytokines11]. Microglia have been shown to express multiple P2 receptorubtypes, including P2X7 and P2Y6 [16]. The P2X7R is a non-elective cation channel and is well-known that P2X7R is involvedn various CNS pathologies including ischemia [7] and neuropathicain [13], via regulating the expression and release of inflam-atory mediators. Moreover, the activation of microglial P2X7

eceptor by endogenous ATP has been associated to the develop-ent of inflammatory responses [21] and neurons sensitization.

urthermore, hypothalamic paraventricular nucleus oxytocinergicnd vasopressinergic neurons modulate cardiovascular responsesn myocardial infarction rats via regulating sympatho-excitatoryction [23,25].

In our present study, we confirmed our hypothesis thatctivation of microglial P2X7R by endogenous ATP promote proin-ammatory cytokines (such as IL-1� and TNF-�) production in theVN, then the activated oxytocinergic and vasopressinergic neu-ons modulate sympatho-excitatory action. These findings wouldffer potential target for AMI therapy.

. Materials and methods

.1. Animal

Adult male Sprague–Dawley rats weighing 200–250 g werebtained from Bikai Sprague–Dawley Inc. They were housed in

temperature-controlled room (22 ± 1 ◦C) with a 12 h light–darkycle.

.2. Coronary ligation, cannula implantation and drugdministration

Animals received intraperitoneal (i.p) administration of the2X7R antagonist Brilliant Blue-G (BBG, 25 or 50 mg kg−1 per dayor 5 days) before surgery [4] and then they were killed after 4 horonary artery ligation. Other animals received bilateral microin-

ig. 1. Laser confocal immunofluorescent view of P2X7R expression in the PVN. Double

euron marker NeuN (B) and two merged image (C and F). Scale bar, 50 �m.

ters 587 (2015) 22–28 23

jection of siRNA (0.015 or 0.03 nmol 0.1 �l per side, once a day for2 days) targeting P2X7R mRNA into the rat PVN [3] and then theywere killed after 4 h coronary artery ligation.

2.3. Tissue microdissection

Brain tissue was quickly removed and cut into 500 �m coro-nal sections. A punch biopsy was obtained from right and left PVNand from right and left peripheral tissue (in the same section)using a 10-gage needle stub (ID: 1.0 mm) according to a previouslydescribed method [21].

2.4. Analysis of mRNA expression by RT-PCR

P2X7R, IL-1�, TNF-� mRNA expression were determinedby RT-PCR as our previously described [14] and Yu et al.report [27]. The primer sequences used for PCR were asfollows: P2X7R forward 5′-CGGGCCACAACTATACTACGA-3′, P2X7R reverse 5′-CCTGAA CTGCCACCTCTGTAA-3′;IL-1� forward 5′-AGCATCCAGCTTCAAATCTCA-3′, IL-1�reverse 5′-CACACTAGCAGGTCGTCATCA-3′; TNF-� forward5′-AGCAGATGGGCTGTACC TTATC-3′, TNF-� reverse 5′-AGGCTGACTTTCTCCTGGTATG-3′; GAPDH forward 5′-CCCTTCATTGACCTCAACTACATG-3′, GAPDH reverse 5′-CTTCTCCATGGTGGTGAAGAC-3′. Gene expression levels of P2X7R, IL-1�,TNF-� mRNA were normalized to GAPDH levels.

2.5. Determination of ATP levels with bioluminescence assay

The level of ATP in the PVN was determined using the ATPbioluminescence assay kit (Beyotime Insititute of Biotechnology,Haimen, China).

2.6. Western blot analysis

Tissue samples of the PVN were subject to western blot analysisas before [24].

2.7. Immunohistochemistry and immunofluorescence

Immunohistochemical studies were performed as our previousreport [24] for vasopressin, oxytocin. A double-staining protocol

immunofluorescence for P2X7R (A and D), the microglia cell marker Iba-1 (E), the

2 ce Letters 587 (2015) 22–28

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Fig. 2. Changes of P2X7R expression and ATP levels in the PVN of different ischemiatime groups. (A) P2X7R protein levels were up-regulated in time-dependent manner,

4 D. Du et al. / Neuroscien

as used for microglial marker Iba-1 (1:400; Abcam) or Neuronarker NeuN (1:300; Abcam) plus P2X7R (1:200; Abcam) staining

n the PVN.

.8. Mean arterial pressure measurement

The femoral artery cannula was flushed with 0.1 ml heparinizedaline (50 U mL−1) and connected to a pressure transducer attachedo a digital blood pressure (BP) monitor and a polygraph. Meanrterial pressure (MAP), intraventricular pressure (IVP), left ven-ricular end-diastolic pressure (LVEDP) and heart rate (HR) dataere collected for 30 min.

.9. Recording and power spectral analysis of heart rateariability (HRV)

HRV was detected as our previous report [14].

.10. RSNA measurement

Basal nerve activity (baseline) was determined by efferent RSNAt the beginning of the experiment. The RSNA activity during thexperiment was calculated by subtracting the background noiserom the recorded value. BBG (i.p) and P2X7 siRNA into the PVN

ere pretreated prior to the acute MI and the RSNA responses werexpressed as the percentage change from the basal value.

.11. Statistical analysis

All of the data are expressed as mean ± SEM. The significance ofifferences in mean values was analyzed by unpaired t-test. Differ-nces were considered significant at P < 0.05.

. Results

.1. Co-localization of P2X7R with Iba-1 or NeuN in the PVN

Double-staining immunohistochemistry was performed in ourxperiments. P2X7R was seen as red fluorescence, microglia markerba-1 and neuron marker NeuN were seen as green fluorescenceFig. 1). We observed that the presence of P2X7R-IR throughouthe confines of the PVN were extensive, and P2X7R-IR were almosto-localized with microglia. Meanwhile, double immunolabelingith P2X7R and Iba-1 or NeuN showed the P2X7R was expressed

n microglia rather than neuron.

.2. ATP levels and P2X7R expression change in the PVN atifferent myocardial ischemia time points

We detected the ATP levels at different ischemia time pointssing bioluminescence assay. The results indicated that the lev-ls of ATP in the PVN was significantly increased during the 8 hyocardial ischemia period and reached the peak at 4 h. Further-ore, the P2X7R protein expression was similar with ATP release

n a time-dependent manner (Fig. 2).

.3. Effects of BBG and P2X7R-siRNA on the expression ofnflammatory mediators and P2X7R in the PVN

P2X7R, IL-1� and TNF-� mRNA and protein were greater inhe PVN of AMI, AMI + BBG and AMI + P2X7-siRNA groups than the

HAM group. Moreover, they were less in the PVN of AMI + BBGnd AMI + P2X7-siRNA groups than in AMI group. There were noifferences among treatment groups on P2X7R, IL-1� and TNF-�xpression in hypothalamic regions adjacent to the PVN (Fig. 3).

(B) showed the relative ratios of P2X7R compared with �-actin and (C) showed theATP changes. Myocardial ischemia, myocardial ischemia for 2 h, 4 h, 8 h, 1 d, 2 d, 4 d,8 d. *P < 0.05 vs. the SHAM group.

3.4. Effects of P2X7R-siRNA on the expression of oxytocin andvasopressin in the PVN

Using immunohistochemistry, we measured oxytocin andvasopressin expression in the PVN of the AMI group and theAMI + siRNA group. The number of oxytocin-IR neurons in the PVNof AMI + siRNA group was significantly less than that of AMI controlgroup (31 ± 3.2 vs. 13 ± 3.5; Fig. 4a). The ROD of oxytocin expressionwas less in AMI + siRNA group than in AMI control group (6.0 ± 0.49vs. 2.3 ± 0.38; Fig. 4a). The number of vasopressin-IR neurons in thePVN of AMI + siRNA group was less than that of AMI control group(25.3 ± 2.4 vs. 8 ± 1.7; Fig. 4b). The ROD of vasopressin expressionwas less in AMI + siRNA group than in AMI control group (4.2 ± 0.26vs. 1.5 ± 0.23; Fig. 4b).

3.5. Effects of P2X7R-siRNA and BBG on RSNA

−1

Intraperitoneal administration BBG (50 mg kg , once a day for5 days) or P2X7R-siRNA (0.03 nmol 0.1 �l per side, once a day for2 days) microinjected into the PVN were performed before themyocardial ischemia and the RSNA responses were expressed as the

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ercentage change from the basal value. As Fig. 5 shown, comparedith the AMI group, the RSNA responses decreased in AMI + P2X7

iRNA and AMI + BBG groups.

.6. Effects of P2X7R-siRNA and BBG on cardiac function and HRV

MAP, HR, IVP and LVEDP were dramatically (P < 0.05) increasedn AMI rats compared with the baseline in SHAM rats and theF/HF ratio was also significantly raised. Compared with AMI rats,

ig. 3. Quantitative comparison of mRNA expression (A) and protein levels (B) for P2X7Rroup. Representative Western blots of P2X7R, TNF-�, IL-1� and �-actin are shown in B. *Pegion.

ters 587 (2015) 22–28 25

both PVN microinjection of P2X7R-siRNA and administration (i.p)of P2X7R antagonist BBG in the AMI + treated rats decreased MAP,HR, IVP and LVEDP, and also descented the LF/HF ratio (Table. 1).

4. Discussion

The adenosine triphosphate (ATP) plays important roles underphysiological and pathological conditions [8] and vesicular ATP canbe released rapidly from microglia through lysosomal exocytosis

, TNF-� and IL-1� from the PVN and regions adjacent to the PVN of each treatment < 0.05 vs. SHAM in the same region; #P < 0.05, AMI + treatment vs. AMI in the same

26 D. Du et al. / Neuroscience Letters 587 (2015) 22–28

Fig. 4. Expression of oxytocin-IR and vasopressin-IR neurons in the PVN of the AMI control group and the AMI + siRNA group. A (×100), B (×200), C (×400): the AMI controlgroup; D (×100), E (×200), F (×400): the AMI + siRNA group. *P < 0.05 vs. the AMI control group. Scale bar, 50 �m.

D. Du et al. / Neuroscience Letters 587 (2015) 22–28 27

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ig. 5. RSNA responses to BBG and P2X7R-siRNA. Segments of original recordings frohanges in RSNA after microinjection of siRNA into the PVN or intraperitoneal injecroup vs. AMI group.

1]. Meanwhile, emerging evidences [19,22] demonstrated that the2X7 receptor, one of the ionotropic P2X receptors for extracel-

ular ATP, played a pivotal roles in the maturation and release ofytokine such as IL-1� and TNF-�. In our study, we found that theelease of ATP increase rapidly in the PVN after myocardial ischemia

n rats. Here, lysosomal exocytosis pathway may be mediated the

icroglial ATP formation and release in the PVN.One novel finding in our study is that the up-regulative

icroglia P2X7 receptor elevated the level of TNF-�, IL-1� in

able 1ardiac function and HRV measurements.

SHAM AMI + V

HR, bpm 378 ± 10 414 ± 1MAP, mm Hg 92 ± 4.62 110 ± 6IVP, mm Hg 21 ± 4.3 49 ± 5LVEDP, mm Hg 2.4 ± 0.25 12.7 ± 2LF/HF 0.22 ± 0.03 0.43 ± 0

ll values are expressed as mean mean ± SEM. AMI + V indicates vehicle-treated AMI; AM* P < 0.05 vs. SHAM.

** P < 0.05, AMI + treated vs. AMI + V. n = 7.

ividual rats from each experimental group show the responses of RSNA (A). (B) meanf BBG. Vehicle indicates aSCF. *P < 0.05 vs. SHAM group; #P < 0.05, AMI + treatment

the PVN and heart rate variability (HRV, indicates sympatheticnerve excitation clinically). This result demonstrate that microglialP2X7 receptor in the PVN contributes to the augmented sympa-thetic nerve activity in acute myocardial ischemic rats. A possibleinterpretation is the microglial P2X7R provide a crucial link

between peripheral sympathetic excitation and central inflamma-tion.

In our study, we observed that P2X7 receptor (co-expressedwith microglia marker Iba-1) was mainly co-located with microglia

AMI + si-RNA AMI + BBG

2* 386 ± 14** 382 ± 11*,**

.08* 94 ± 8.08** 90 ± 6.06**

.2* 24 ± 2.6** 34 ± 3.0*,**

.4* 8.3 ± 0.76*,** 7.9 ± 0.89*,**

.04*,** 0.30 ± 0.04*,** 0.29 ± 0.05*,**

I + siRNA, P2X7-siRNA treated AMI; AMI + BBG, P2X7R antagonist BBG-treated AMI.

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ather than neurons in PVN and the P2X7R/Iba-1 positive cells werenalyzed. Furthermore, the P2X7 protein increased rapidly dur-ng the early phases of myocardial ischemia. However, after 8 h

yocardial ischemia, the expression of P2X7 protein in the PVNrend to decrease. There is one possible reason for interpreting thisesult, acute myocardial ischemia induce production of ATP thatctivate P2X7 receptor in the PVN. Our results appear consistentith the previous studies microglial cells, which could be activated

nder conditions of brain injury or inflammation [21], can produce wealth of inflammatory mediators, including the proinflamma-ory cytokines, TNF-� and IL-1� [2]. Therefore, the expression ofroinflammatory cytokines in the PVN of AMI rats could be regu-

ated by microglial P2X7 receptor inside the BBB. These results wereonsistent with the previous report that the production of proin-ammatory cytokines was related to the microglial P2X7 receptor

6].In addition, one other new finding of this study is: ATP-induced

icroglial P2X7 receptor activation enhanced the expression ofNF-� and IL-1�, which may activated the neurons in the PVN. Fur-hermore, which kinds of neurons were activated in the process?ur results found that oxytocin and vasopressin were overex-ressed in the PVN of AMI rats and this overexpression cane suppressed by microinjection of P2X7R-siRNA into the PVN.ogether, these results indicated that oxytocinergic and vaso-ressinergic neuron may be participated in regulating the acuteyocardial infraction. Related reports [25,26] confirmed oxytocin-

rgic and vasopressin neuron regulate sympatho-excitatory action.eanwhile, we found that administration of BBG, a noncompet-

tive P2X7 receptor antagonist, or P2X7R knockdown in the PVNrevented the rise of renal sympathetic nervous activities (RSNA)

n the AMI rats. Clinical studies have shown that analysis of HRVrovides information about the disturbances of the cardiac auto-omic nervous system. Therefore, we use LF/HF ratio to indicatehe SNA.

The significance of the production of proinflammatory cytokinesn PVN microglia is presently unknown. Nevertheless, cytokinesre the potent stimuli for activation of the nuclear tran-cription factor kappa B (NF-�B) [20], which can produce aariety of excitatory mediators/inflammatory, including glutamateGlu)/norepinephrine (NE) and tyrosine hydroxylase (TH) [15],ithin the brain parenchyma. Furthermore, NF-�B also regulate

he activities of angiotensin II type 1 receptor (AT1-R), superoxidend gp91 (a subunit of NADP(H) oxidase) [17,15] in many kinds ofathological conditions. Therefore, additional studies are neededo determine whether NF-�B activation in AMI rats induces thexpression of the AT1-R and superoxide [10] that might activate theympathetic nervous system in these disease processes. Other pointeserve comment, studies of P2X7 receptor functions in animal typ-

cally use BBG 25–50 mg kg−1 per day, but this dose is impossibleo apply clinically.

In summary, the activated microglial P2X7 receptor induced byTP promote the TNF-� and IL-1� release in the PVN, then theNF-� and IL-1� could activate oxytocinergic and vasopressin neu-on that regulate the renal sympathetic nerve activity. And thenhe sympathetic nerve regulate the cardiac function in the acute

yocardial infarction rat. Manipulations designed to inhibit P2X7Rctivation in the PVN may be effective method to the current treat-ent of AMI.

cknowledgment

This work was supported by National Natural Science Founda-ion of China (No. 31100838, No. 31271215, No. 31172147).

[

ters 587 (2015) 22–28

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