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Neuroscience Letters 509 (2012) 125– 129

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Neuroscience Letters

jou rn al h om epage: www.elsev ier .com/ locate /neule t

Lateral hypothalamic area mediated the aggravated effect of microinjection ofBaclofen into cerebellar fastigial nucleus on stress gastric mucosal damage in rats

Jin-Zhou Zhua,b,1, Su-Juan Feia,1, Jian-Fu Zhanga,b,∗, Sheng-Ping Zhua,b, Zhang-Bo Liua,b,Ting-Ting Lia,b, Xiao Qiaoa,b

a Department of Gastroenterology, Affiliated Hospital of Xuzhou Medical College, 99 West Huaihai Road, Xuzhou, 221002 Jiangsu, Chinab Department of Physiology, Xuzhou Medical College, 84 West Huaihai Road, Xuzhou, 221002 Jiangsu, China

a r t i c l e i n f o

Article history:Received 3 November 2011Received in revised form27 December 2011Accepted 28 December 2011

Keywords:BaclofenCerebellumFastigial nucleusGamma-aminobutyric acidLateral hypothalamic areaStress gastric mucosal damage

a b s t r a c t

Cerebellum, primarily believed as a subcortical somatic motor center, is increasingly considered to beimplicated in visceral activities. However, little is known about its regulation on gastrointestinal organs.In this research, we investigated the aggravated effect of microinjection of gamma-aminobutyric acidreceptor subtype B (GABABR) agonist, Baclofen into cerebellar fastigial nucleus (FN) on stress gastricmucosal damage (SGMD) and its possible regulatory mechanism. The gastric mucosal damage indexwas chosen to indicate the severity of gastric mucosal injure. Immunohistochemistry and transferase-mediated dUTP-biotin nick-endlabeling (TUNEL) methods were used to detect the variations of lateralhypothalamic area (LHA) and gastric mucosa. It had been demonstrated that FN participates in regulationof SGMD via its GABABR and GABA neural pathway, which passes through the decussation of superiorcerebellar peduncle and projects to the GABA receptors in LHA. Meanwhile, celiac sympathetic nerveinvolves in this process via mediating neural discharge, which results in the decrease of gastric mucosalblood flow. Additionally, apoptosis, proliferation and oxidation in gastric mucosa, and gastric acid con-tribute in the mechanism. It could be expected that these results might suggest insights to the cerebellarand hypothalamic function, and the treatment of gastrointestinal diseases.

© 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

As is known, cerebellum, a traditional considered subcorticalsomatic motor center, also is an essential component in the centralintegration of visceral activities [17]. However, little is known aboutits regulation on gastrointestinal organs.

In the previous work [9], we presumed that cerebellar fastigialnucleus (FN) neurons involve in the regulation of stress gastricmucosal damage (SGMD), but the detail of its possible mecha-nism was undetected. Numerous data had demonstrated that deepcerebellar nuclei (include FN, Int and Lat) neurons are predomi-nantly controlled by gamma-aminobutyric acid (GABA) inhibitory

Abbreviations: CSN, celiac sympathetic nerve; DSCP, decussation of superiorcerebellar peduncle; FN, fastigial nucleus; GMBF, gastric mucosal blood flow; GMDI,gastric mucosal damage index; LHA, lateral hypothalamic area; RWI, restraintand water (21 ± 1 ◦C)-immersion; SGMD, stress gastric mucosal damage; TUNEL,transferase-mediated dUTP-biotin nick-endlabeling.

∗ Corresponding author at: Xuzhou Medical College, 84 West Huaihai Road,Xuzhou, 221002 Jiangsu, China. Tel.: +86 516 8562 6128; fax: +86 516 8558 2071.

E-mail address: jfzhang@xzmc.edu.cn (J.-F. Zhang).1 Both authors contributed equally to this work.

neurons from cerebellar cortex [19,22]. Furthermore, someresearch revealed that GABA receptor subtype B (GABABR) presentsin FN [23]. Thus, we suspected that GABABR in FN possibly is impli-cated in the regulation of cerebellum on SGMD.

Increasing works supported that the neuronic axons of FNprimarily pass through the decussation of superior cerebellarpeduncle (DSCP) and project to the GABA receptors in lateralhypothalamic area (LHA), which mainly include GABA receptorsubtype A subunit �1 (GABAAR�1) and GABA receptor subtypeB subunit 1 (GABABR1) [2,3,11]. In the present research, we alsotried to reveal the character of LHA in the mediative mecha-nism.

As demonstrated previously, LHA links to the parasympatheticand sympathetic nerves [13]. Our former studies showed thatparasympathetic and sympathetic nerves involve in the regulatoryprocess of LHA or paraventricular nucleus (PVN) on some gastricactivities [25,26]. The integrity of gastric mucosa is maintainedby the balance of apoptosis and proliferation in gastric mucosalepithelium [14]. And many reports also suggested the possiblecontributions of gastric gland secretory function and oxidation onSGMD [7,15]. Accordingly, it is imperative to discover the charac-ters of visceral nerves and the variations of gastric mucosa in thisregulation.

0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.neulet.2011.12.057

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Fig. 1. Effect of microinjection of Baclofen into FN on SGMD and roles of DSCP, LHA and PVN. (A): effect of microinjection of Baclofen into FN on SGMD. Normal: nor-mal; RWI: restraint and water (21 ± 1 ◦C)-immersion, only; Vehicle: microinjection of Vehicle + RWI; B. & C.: A cocktail of Baclofen and CGP55845 was microinjected intoFN + RWI; 2.50 �g, 5.00 �g, 10.00 �g: different doses of Baclofen were microinjected into FN individually + RWI. Mean ± SEM; n = 6; *P > 0.05, vs Vehicle group; **P < 0.01, vsVehicle group. (B) and (C): roles of DSCP, LHA and PVN in the effect. SDA + V + RWI: sham DSCP ablation + Vehicle + RWI; SDA + B + RWI: sham DSCP ablation + Baclofen + RWI;DA + B + RWI: DSCP ablation + Baclofen + RWI; SLA + V + RWI: sham LHA ablation + Vehicle + RWI; SLA + B + RWI: sham LHA ablation + Baclofen + RWI; LA + B + RWI: LHA abla-tion + Baclofen + RWI; SPA + V + RWI: sham PVN ablation + Vehicle + RWI; SPA + B + RWI: sham PVN ablation + Baclofen + RWI; PA + B + RWI: PVN ablation + Baclofen + RWI.Mean ± SEM; n = 6; #P < 0.01 vs SDA + B + RWI group or SLA + B + RWI group; ##P > 0.05 vs SPA + B + RWI group.

2. Materials and methods

Adult male Sprague-Dawley (SD) rats, weighing 210–230 g,were provided by Experimental Animal Center of Xuzhou Medi-cal College. All experiments were performed according to the U.S.National Institute of Health Guide for the Care and Use of Labo-ratory Animals. The animals were divided randomly to differentgroups (n = 6). The room temperature was kept at 23 ± 1 ◦C undera 12/12 h day/night period circle. Before the experiments, all ratswere fasted for 24 h, but allowed to drink water. At the end, therats were anesthetized with overdose of sodium pentobarbital. Therats’ stomachs and brains were removed rapidly and immersed in10% paraformaldehyde for 48 h, and then embedded in paraffin,sliced to 4 �m thick, mounted on glass slides for the immunohisto-chemistry and transferase-mediated dUTP-biotin nick-endlabeling(TUNEL) assays.

The rats were anesthetized hypodermically with sodium pento-barbital (40 mg/kg), and then mounted on a stereotaxic apparatus.The scalp was incised, and then a hole, 0.5 mm in diameter, wasdrilled above the cranium dorsal of the target site. The coordinates

for the FN, DSCP, LHA and PVN were determined in accordance withthe rat brain atlas in Stereotaxic Coordinates [21].

Baclofen, GABABR agonist (Sigma–Aldrich, USA), was microin-jected into the paired FN through a microsyringe. The standarddose of Baclofen is 5.00 �g in 0.3 �l 0.9% saline in Baclofen injectiongroups [12], nevertheless, we used three different doses (2.50, 5.00,10.00 �g in 0.3 �l 0.9% saline) to detect the correlation of doses andeffect. The injection lasted for 2 min and the microsyringe was leftin the positions for another 3 min to prevent backflow. The vehicle(0.3 �l 0.9% saline) was microinjected into paired FN in Vehiclegroups and sham ablation groups individually. In B. & C. group, wemade a 0.3 �l cocktail of Baclofen (5.00 �g) and CGP55845, GABABRantagonist (5.00 �g, Sigma–Aldrich, USA), microinjected into FNto certify that the effect was associated with GABABR specifically[12]. Electrical ablation of DSCP [9] was executed by passing apositive DC current of 1 mA for 10 s. Sham DSCP ablation groupwere accomplished as the same procedure above, but no currentwas passed. Ablations of LHA or PVN were made with the microin-jection of Kainic acid (0.30 �g in 0.3 �l saline, Sigma–Aldrich,USA) into the paired LHA and PVN [4]. DSCP, PVN and LHA were

Fig. 2. Effects of microinjection of Baclofen into FN on the number of GABA positive neurons and the expressions of GABAAR�1 and GABABR1 in LHA. (A) and (B): the variousnumbers of GABA positive neurons in LHA. (C) and (D): the variation of the expression of GABAAR�1 in LHA. (E) and (F): the variation of the expression of GABABR1 in LHA.Normal: normal; Baclofen: microinjection of Baclofen into FN. The sections pointed by the arrows are LHA (scale bar: 200 �m). Mean ± SEM, n = 6; *P < 0.05 vs Normal group.

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destructed electrically or chemically 3 days before the microin-jection and induction of restraint and water (21 ± 1 ◦C)-immersion(RWI). The model of SGMD and gastric mucosal damage index(GMDI) were performed according to the method described [9].

For the LHA immunohistochemistry, the rats had been microin-jected into FN with Baclofen 1 time per day for 3 days. The numberof GABA positive neurons and the expressions of GABAAR�1 andGABABR1 were assessed by immunohistochemical method, usingan SABC Rabbit IgG POD Kit (Boster, China) on paraffin slices,according to the manufacture instruction and the previous work[10]. Rabbit anti-rat GABA, GABAAR�1 and GABABR1 antibodies(diluted to 1:50 in PBS) were used as the primary antibodies.Biotinylated goat anti-rabbit IgG was the bridging antibody (Boster,China). The primary antibodies were replaced by PBS for negativecontrol. Quantitative analysis was performed via measuring inte-grated optical density (IOD) of LHA sections in 3 inconsecutive slicesfrom each specimen [5].

Celiac sympathectomy, truncal vagotomy and sham operationswere performed as described before respectively [1,18]. Before themicroinjection, sympathectomy, vagotomy and sham operationswere performed 1 week ago. The measurement of gastric mucosalblood flow (GMBF) and discharge of celiac sympathetic nerve (CSN)were performed as reported before [9,20].

The expression of proliferating cell nuclear antigen (PCNA) weredetected by immunohistochemical method as the above descrip-tion and previous study [16]. Rabbit anti-PCNA (diluted to 1:100in PBS) and biotinylated goat anti-rabbit IgG antibody were chosenas primary and bridging antibody (Boster, China). Eventually, theaverage numbers of proliferative cells were collected from 3 incon-secutive slices in each specimen. And quantitative analysis wasaccomplished via counting randomly the numbers of PCNA posi-tive cells in ten microscopic fields. TUNEL staining was performedon paraffin slices, using an in situ cell death detection kit (RocheDiagnostics, Germany) and referring to instruction of manufactureand previous study [24].

The measurement of malondialdehyde (MDA) and superox-ide dismutase (SOD) was performed as reported [8]. Gastric juicevolume and acidity were measured in accordance with the previ-ous work [6]. The variation of the gastric acid secretion had beenobserved for 3 h after the microinjection.

All results were expressed as mean ± SEM. Comparisonsbetween two groups were made using Student’s t-test, multiple-group analyses were made by one-way ANOVA test. Statisticalanalysis was performed with GraphPad Prism 5 and SPSS 16.0. Theresults were considered significantly different at P < 0.05.

3. Results

As indicated in Fig. 1A, GMDI was 52.17 ± 2.71 in RWI group and50.17 ± 3.28 in Vehicle group, whereas no damage was observed inNormal group. Compared with Vehicle group, B. & C. group was50.83 ± 3.94 (P > 0.05). However, GMDI were obviously increasedindividually (68.33 ± 2.59, 78.50 ± 3.64, 92.67 ± 3.53, P < 0.01) aftermicroinjection of different doses of Baclofen into FN and RWI. Addi-tionally, a statistical analysis had been made about the correlationof the baclofen dose and GMDI, which showed that it was significantat the 0.01 level.

To ascertain whether DSCP, LHA and PVN involve in the reg-ulation, Fig. 1B showed that the GMDI was 77.83 ± 4.36 in shamDSCP ablation + Baclofen + RWI group, subsequently reduced to53.50 ± 3.87 (P < 0.01) in DSCP ablation + Baclofen + RWI group. InFig. 1C, It was 81.00 ± 3.13 in Sham LHA ablation + Baclofen + RWIgroup, and then dropped to 53.01 ± 2.94 (P < 0.01) in LHA abla-tion + Baclofen + RWI group. However, there was no notabledifference (P > 0.05) between Sham PVN ablation + Baclofen + RWI

Fig. 3. Roles of celiac sympathetic and vagus nerves in effect of microinjection ofBaclofen into FN on SGMD and the variations of GMBF. (A): roles of sympatheticand vagus nerves in the effect. SS + V + RWI: sham sympathectomy + Vehicle + RWI;SS + B + RWI: sham sympathectomy + Baclofen + RWI; Sym + B + RWI: sympathec-tomy + Baclofen + RWI; SV + V + RWI: sham vagotomy + Vehicle + RWI; SV + B + RWI:sham vagotomy + Baclofen + RWI; Vag + B + RWI: vagotomy + Baclofen + RWI.Mean ± SEM, n = 6; *P < 0.01 vs SS + B + RWI group; **P > 0.05 vs SV + B + RWI group.(B) and (C): the effect on the discharge frequency and intensity of CSN. (D): theeffect on GMBF. Normal: normal; RWI: restraint and water (21 ± 1 ◦C)-immersion,only; Vehicle: microinjection of Vehicle into FN + RWI; Baclofen: microinjection ofBaclofen into FN + RWI. Mean ± SEM, n = 6; #P < 0.01 vs Normal group; ##P < 0.05 vsVehicle group.

group and PVN ablation + Baclofen + RWI group. Fig. 2A and Brevealed that the IOD of LHA sections declined in Baclofen group,compared with Normal group (P < 0.05). Meanwhile, the samechange happened to the expressions of GABAAR�1 (Fig. 2C and D)and GABABR1 (Fig. 2E and F) too.

In Fig. 3A, the GMDI was 82.63 ± 5.13 in sham sympathec-tomy + Baclofen + RWI group, whereas, it turned to 51.17 ± 4.02(P < 0.01) in sympathectomy + Baclofen + RWI group. Interesting,GMDI was 84.17 ± 5.46 in vagotomy + Baclofen + RWI group, whichwas no significant difference with sham vagotomy + Baclofen + RWIgroup (81.99 ± 5.15, P > 0.05). And CSN mean discharge frequencyand intensity increased after the microinjection (P < 0.01) (Fig. 3B

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Fig. 4. Effects of microinjection of Baclofen into FN on proliferation, apoptosis and oxidation of gastric mucosa on SGMD. Normal: normal; RWI: restraint and water (21 ± 1 ◦C)-immersion, only; Vehicle: microinjection of Vehicle into FN + RWI; Baclofen: microinjection of Baclofen into FN + RWI. (A) and (B): the variations of the proliferation in gastricmucosal cells. (C) and (D): the variations of the apoptosis. Scale bar: 20 �m. Mean ± SEM; n = 6; *P < 0.01, vs Normal group; **P < 0.01, vs Vehicle group. E & F: the effects onMDA content and SOD activity. Mean ± SEM, n = 6, #P < 0.01 vs Normal group, ##P < 0.01 vs Vehicle group.

and C). Additionally, GMBF showed a notable decrease in Baclofengroup, compared with Vehicle group (P < 0.05) (Fig. 3D).

In Fig. 4A and B, the percentage of proliferative cells (brown)in Normal group was 50.33 ± 5.20%, whereas it decreased from23.33 ± 1.92% and 23.83 ± 1.99% in RWI and Vehicle groupsto 10.33 ± 2.01% (P < 0.01) in Baclofen group. Fig. 4C and Dshowed that although the percentage of apoptotic cells (brown)was 3.90 ± 1.03% in Normal group, whereas compared withRWI and Vehicle groups (42.67 ± 3.45% and 44.33 ± 3.06%),a remarkable growth had been observed in Baclofen group(70.67 ± 3.06%, P < 0.01). Additionally, the MDA content of gas-tric mucosa was markedly increased in the Baclofen group(11.89 ± 0.48 nmol/mg), compared with RWI and Vehicle groups(7.33 ± 0.46 and 7.32 ± 0.49 nmol/mg, P < 0.01) (Fig. 4E), although itwas 4.39 ± 0.43 nmol/mg in Normal group. And, the SOD activityin Normal group was 104.5 ± 6.45 U/mg, and in RWI and Vehi-cle groups were 75.17 ± 6.19 and 75.83 ± 5.73 U/mg, eventually itdropped to 36.33 ± 4.35 U/mg in Baclofen group (P < 0.01). Table 1indicated that the microinjection of Baclofen enhanced the gastricjuice volume and acidity.

4. Discussion

At current research, it had been observed that the microin-jection of Baclofen into FN obviously intensified SGMD in adose-dependent manner. Additionally, the microinjection witha cocktail of Baclofen and CGP55845 into FN failed to exert an

Table 1Effect of microinjection of Baclofen into FN on gastric juice volume and acidity inrats.

Normal (n = 6) Baclofen (n = 6)

Volume (ml) 1.62 ± 0.17 3.21 ± 0.36*

Acidity (10−5mol/ml) 5.01 ± 0.34 8.32 ± 1.79*

Figures in (n =) indicate the numbers of rats. Each value represents with mean ± SEM.* P < 0.05 vs Normal group.

influence on SGMD. Consequently, the GABABR is one of the vitalcomponents in the regulation of FN on SGMD.

The effect of microinjection of Baclofen on SGMD was invalidwith the pretreatment of electrical lesion of DSCP and chemicalablation of LHA individually, not ablation of PVN. And the numberof GABA positive neurons and the expressions of GABAAR�1 andGABABR1 in LHA decreased after the microinjection of Baclofen intoFN. We believed that microinjection of Baclofen into FN suppressedthe neural activity of FN that reduced the synthesis and releaseof the inhibitory neurotransmitters, GABA. And through DSCP, thereduced GABA neurotransmitters integrated with GABA-A and -B receptors in LHA. The down-regulation of inhibitive integrationresulted in the up-regulation of LHA neural activity.

Meanwhile, the effect of microinjection of Baclofen was can-celled by the pretreatment of celiac sympathectomy, however, notvagotomy. Besides, we witnessed the promotion of frequency andintensity in the discharge of CSN and the decrease of GMBF. Pre-sumably, microinjection of Baclofen increased the discharge of CSNmediated by LHA, which resulted in the vasoconstriction in gastricmucosa, thus, intensified SGMD.

SGMD is a multifaceted pathological process. Our study demon-strated that the microinjection of Baclofen into FN intensifiedSGMD in rats via the promotion of gastric mucosal cellular apo-ptosis and the inhibition of proliferation. Besides, anti-oxidationstatus in gastric mucosa decreased, and the volume and acidity ofgastric juice increased after the microinjection of Baclofen.

In conclusion, it could be expected that our findings suggesta novel understanding of the cerebellar and hypothalamic func-tions, and a possible approach to the treatment of gastrointestinaldiseases.

Acknowledgements

This project was supported by grants from National NaturalScience Foundation of China (No. 30570671), Educational Sci-ence Research Foundation of Jiangsu Province (No. 10KJB310015),

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Research Foundation of Xuzhou Medical College (Nos. 07KJ58 and07KJ34) and Xuzhou Social Development Fund (No. XM08C062).

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