EXPRESSION OF c-fos IN THE MEDULLA OBLONGATA
AFTER CAROTID BARORECEPTOR ACTIVATION BY
ELEVATED INTRASINUS PRESSURE AND ADENOSINE

CHEN　SHUANG，ZHANG　XIAO-XUE^*,SHI　GE-MING^*,HE　RUI-RONG^**
(Departments of Physiology and ^*Anatomy, Hebei Medical University, Shijiazhuang 050017)

ABSTRACT　　 Expression of c-fos protein in the medulla oblongata after baroreceptor activation by elevated intrasinus pressure (ISP) and perfusion of adenosine (Ado) was examined in 14 vascularly isolated carotid sinus perfusion rats. The results showed that Fos-like immunoreactive(FLI) neurons were distributed throughout nucleus tractus solitarius, area postrema, rostral ventrolateral medulla and nucleus raphe pallidus, and the number of FLI was increased with the elevation of ISP. Furthermore, perfusing the carotid sinus with Ado at a given ISP markedly increased the FLI in the above regions. From the results obtained, it is concluded that the c-fos expression in baroreflex pathway in medulla oblongata may be enhanced by elevated ISP and intrasinus perfusion of Ado, and Ado is capable of facilitating the baroreflex.
Key words:c-fos; intrasinus pressure; adenosine

窦内压升高和灌流腺苷激活颈动脉窦
压力感受器时延髓内c-fos蛋白的表达

陈　爽　张小雪^*　石葛明^*　何瑞荣^**

摘　　要

　　在14 只隔离灌流颈动脉窦区的大鼠, 观察了窦内压(ISP) 升高和灌流腺苷 (adenosine, Ado) 激活压力感受器时延髓内c-fos蛋白的表达。 结果显示: 在孤束核、 最后区、 延髓腹外侧头端区和中缝苍白核可见Fos蛋白样免疫阳性反应（FLI）神经元分布, 且其数量随ISP升高而增多。 在给定ISP下, 颈动脉窦内灌流Ado, 可使上述区域中FLI表达明显增多。 根据以上结果, 得出如下结论: c-fos在压力感受器反射延髓通路中的表达, 可由ISP增高和灌流Ado而增强, 表明Ado对压力感受器反射有易化作用。
关键词: c-fos; 窦内压; 腺苷
学科分类号: Q463

　　Expression of Fos-like protein was used as a specific marker for activated central neurons involved in the baroreceptor reflex^[1]. In 1991, Jeffery et al. reported that direct electrical stimulation of carotid sinus nerve (CSN) in anesthetized rats resulted in a consistent and discrete distribution of Fos-like immunoreactive (FLI) neurons within the medulla oblongata^[2]. McKitrick et al. observed that electrical stimulation of depressor nerve in rats might induce c-fos expression in nucleus tractus solitarius (NTS), area postrema (AP) and ventrolateral medulla (VLM)^[3]. Repeated and scheduled activation of the baroreceptor by transient hypertension induced by i.v. administration of phenylephrine elicited a significant increase of FLI in NTS^[4,5] and VLM^[6]. However, the techniques used in these studies have the limitation that baroreceptor was not activated by physiological mode of stimulation. Our previous studies showed that a typical functional curve for intrasinus pressure (ISP)sinus nerve afferent activity relationship might be obtained with very great reproducibility by perfusing carotid sinus in anesthetized rats, and adenosine (Ado) did facilitate carotid baroreflex and increase baroreceptor activity^[7,8]. The present study was aimed to examine the distribution of c-fos immunoreactive neurons in medulla oblongata in response to baroreceptor stimulation by elevating ISP in rats, and to investigate the neurons involved in the baroreflex pathway in medulla oblongata immunohistochemically when the vascularly isolated carotid sinus was perfused with Ado.

1　MATERIALS AND METHODS
　　The experiments were performed on Sprague-Dawley rats weighing 300～350 g. The animals were anesthetized intraperitoneally with urethane (1.0 g/kg). All the animals were taken out from their cages at the same time of the experimental day, and handled as gently as possible. During the experiments, each animal was placed on a soft cushion, and the ambient temperature and environment were kept constant and quiet to avoid undesirable stimuli. The animals breathed spontaneously through an intratracheal tube. The right femoral artery was catheterized for monitoring systemic blood pressure with a pressure transducer (MPU-0.5, Nihon Kohden).
1.1　Carotid sinus perfusion　　 The method of carotid sinus perfusion has been previously described in detail^[8]. In brief, the left carotid sinus was isolated from the systemic circulation and perfused with Krebs-Henseleit (K-H) solution. The perfusate was bubbled with 95% O₂ and 5% CO₂, and warmed in a water bath at 37℃. Using a computer controlling system, the ISP could be adjusted automatically^[9]. The ISP was kept at 13.3 kPa before the carotid baroreflex was examined. To observe the baroreflex response, ISP was raised from 0 to 33.3 kPa in a ramp manner and lasted for 30 s, then regaining the original holding pressure of 13.3 kPa.
1.2　Protocols　　The experimental animals were divided into four groups.Group 1 (n=4)By perfusing carotid sinus with K-H solution, ISP was kept at 6.7 kPa for 90 min, then the ascending aorta was cannulated and perfused with 150 ml of physiological saline solution, followed by 300 ml of 4% paraformaldehyde (pH 7.4). The brains were taken out immediately. This group of rats were used for measuring the level of c-fos expression in low ISP state. Group 2 (n=3)By perfusing carotid sinus with K-H solution, ISP was kept at 16.0 kPa for 1 min, then ISP was lowered to 13.3 kPa and remained for 5 min. This cycle was repeated up to 90 min. The other processes were the same as in group 1. Group 3 (n=4) By perfusing carotid sinus with K-H solution, ISP was kept at 26.7 kPa for 1 min, then lowered to 13.3 kPa and remained for 5 min. This process was repeated up to 90 min, at the end of which the animals were perfused and fixed as in group 1. Group 4 (n=3) The carotid sinus was perfused with K-H solution containing Ado (175 μmol/L). The change of ISP was monitored in the same way as in group 2. At the end of the 90 min of stimulation the animals were perfused and fixed as in group 1.
　　The removed brains were fixed in 4% paraformaldehyde for 4 h, then transferred to a 30% sucrose solution and kept overnight to prevent ice crystal formation. Coronal sections (40 μm) of brains were cut in a cryostat. Every third tissue section was processed for Fos-like protein by the avidin-biotin-complex technique.
1.3　Immunohistochemistry　　The sections were washed three times in phosphate buffer solution (PBS) for 5 min followed by 3% hydrogen peroxide to quench endogenous peroxidase activity. Sections were placed in PBS for 5 min and incubated in normal goat serum (5% in 3% Triton X-100 in PBS) for 1 h. The serum was removed and the sections were then incubated in a primary antibody (1∶500 dilution, Santa Cruz Biotechnology) for 1 h at room temperature, then for 48 h at 4℃. At the end of this incubation period, sections were rinsed three times in PBS for 5 min. The sections were incubated in biotinylated goat anti-rabbit secondary antibody (1∶300 dilution) for 1 h at room temperature, rinsed in PBS for three 5-min and incubated in avidin and biotinylated horseradish peroxidase macromolecular complex diluted 1∶300 for 1 h at room temperature. After three 5-min washes in PBS, the c-fos reaction product was made visible by light microscope. c-fos reaction products appeared as dark brown stainings in the cell nucleus.
1.4　Cell count and statistical analysis　　Tissue sections were examined under standard light microscope. The total number of labeled cells was counted in each region for each animal according to the brain altas of Paxinos and Watson^[10]. All the data were expressed as ±s. The differences in the reflex decrease in BP and Fos-positive neuronal counting between groups were compared by unpaired t-test. Statistical significance was accepted when P<0.05.

2　RESULTS
　　Carotid sinus afferent fibers were activated with high ISP in the rat^[7]. The effectiveness of the stimulation was assessed by reflex decrease in BP^[8].

2.1　Expression of c-fos in medulla oblongata at low ISP
　　As the ISP at 6.7 kPa, the systemic BP was reflexly elevated to high level (Fig.1A). Only a little Fos protein was present in neurons throughout the NTS, AP, RVLM and nucleus raphe pallidus (RP) (Fig.2A, see Plates Ⅰand Ⅱ). The mean numbers of FLI neurons in these areas were 13.5±1.9, 42.6±3.6, 3.5±0.8 and 1.3±0.8, respectively (Fig.3).

Fig.1　Baroreflex response of arterial blood pressure (BP) to the increased intrasinus pressure (ISP)
A. ISP, 6.7 kPa; B. ISP, 16.0 kPa; C. ISP, 26.7 kPa; D. ISP, 16.0 kPa+Ado (175 μmol/L).

2.2　Expression of c-fos in medulla oblongata at middle ISP
　　Reflex decrease of BP (RD) was 1.34±0.01 kPa with ISP at 16.0 kPa (Fig.1B). In 3 animals, labeled cells were present in the same regions as in those of the animals with ISP at 6.7 kPa, while FLI neurons were significantly increased (each B in Fig.2, see Plates Ⅰ and Ⅱ). The numbers of immunopositive cells in NTS, AP, RVLM and RP were 28.2±2.7, 63.2±4.3, 8.9±1.6 and 9.1±1.6, respectively (P<0.001 vs group 1, Fig 3)

Fig.1　Baroreflex response of arterial blood pressure (BP) to the increased intrasinus pressure (ISP)
A. ISP, 6.7 kPa; B. ISP, 16.0 kPa; C. ISP, 26.7 kPa; D. ISP, 16.0 kPa+Ado (175 μmol/L).

2.3　Expression of c-fos in medulla oblongata at high ISP
　　When ISP was kept at 26.7 kPa, RD was 4.1±0.02 kPa (Fig.1C). The immunoreactive cells were significantly increased in the regions mentioned above as compared with those of middle ISP animals (Fig 2C, see Plates Ⅰ and Ⅱ). The mean numbers of labeled nuclei in the high ISP group were higher than those in the middle ISP group (Fig 3).

Fig.1　Baroreflex response of arterial blood pressure (BP) to the increased intrasinus pressure (ISP)
A. ISP, 6.7 kPa; B. ISP, 16.0 kPa; C. ISP, 26.7 kPa; D. ISP, 16.0 kPa+Ado (175 μmol/L).

s2.4　Expression of c-fos in medulla oblongata at middle ISP with perfusion of Ado
　　As carotid sinus was perfused with K-H solution containing Ado (175 μmol/ L) and ISP was held at 16.0 kPa, the RD of this group was more marked than that of group 2 (P<0.05, Fig 1D) with more FLI neurons than those of middle ISP group (P<0.01) (Fig.2D, see Plate Ⅱ, and Fig.3 ).

Fig.1　Baroreflex response of arterial blood pressure (BP) to the increased intrasinus pressure (ISP)
A. ISP, 6.7 kPa; B. ISP, 16.0 kPa; C. ISP, 26.7 kPa; D. ISP, 16.0 kPa+Ado (175 μmol/L).

Fig.3　Histograms showing the mean number of Fos-like immunoreactive (FLI) cells per section in the medulla oblongata after carotid barorecepter activation by increased ISP and adenosin
NTS: nucleus tractus solitarius; AP: area postrema; RVLM: rostral ventrolateral medulla; RP: nucleus raphe pallidus. L. ISP, 6.7 kPa; M. ISP, 16.0 kPa; H. ISP, 26.7 kPa. ^***　P<0.001 vs L, ^+　+　P<0.01, ^+　+　+　P<0.001 vs M.

Fig.2 For legend see the next page

Fig.2 Microphotographs showing the FLI neurons in nucleus tractarius (NTS),area postrema (AP),rostral medulla (RVLM) and nucleus sraphe pallidus (RP) A.ISP, 6.7 kPa:;B.ISP,16.0kPa;C.ISP,26.7kPa;D.ISP,16.0kPa+Ado (175umoI/L)

3　DISCUSSION
　　In the present study, it was demonstrated that the increase of ISP in the vascularly isolated carotid sinus of the rat might induce c-fos expresion in medulla oblongata. FLI was observed bilaterally within NTS, RVLM, area postrema and nucleus raphe pallidus.
　　While the basal expression of Fos is relatively low in central neuronal system^[11], it is transiently expressed in neurons after synaptic stimulation and has been used as a metabolic marker in neuronal tracing^[12]. Baroreceptor afferents project primarily to the neurons in the NTS, and also to other brainstem areas including the AP and RP^[13,14]. Whereas anatomic tracing studies have demonstrated that the AP receives its afferent input from NTS^[15] and the AP also sends dense projections to NTS^[16]. Agarwal et al. demonstrated that cardiovascular neurons in NTS and RVLM are reciprocally connected and involved in reflex loops controlling the baroreceptor reflex^[17]. Our results indicate that the expression of FLI in medulla is induced by barorecepor activation. It is reported that FLI cells may also be expressed in the above-metioned areas following hypoxic stimulation of carotid sinus afferents^[2] or electrical stimulation of the depressor nerve^[3]. In our experiment, bilateral depressor nerves were cut, and the occipital artery was ligated so as to eliminate the activity of aortic baroreceptor and carotid chemoreceptor. So it is reasonable to consider that the significant expression of c-fos in our experiment may be attributed to carotid baroreceptor activation by physiologically increased pressure. The expression level of c-fos varied at different ISP. The higher the ISP was, the more numbers of central barosensitive neurons were activated. Thus, there was a higher level of Fos expression in the high ISP than in the low one. This is consistant with the results of Dean and Seagard observed in NTS of anesthetized dogs^[18]. Operation, handling and stress appear to elevate neuronal Fos levels^[19]. The basal levels being measured at low ISP might be greatly influenced by the past history (1～12 h) of the experimental animals.
　　Our previous study on anesthetized rats indicated that Ado could increase the baroreceptor activity and facilitate baroreflex^[7,8]. The present results indicate that the expression level of c-fos in baroreflex pathway in medulla oblongata induced by perfusing with Ado was higher than that by perfusing K-H solution at the same ISP. Such a result further supports morphologically that Ado facilitates the afferent activity of carotid sinus in anesthetized rats.
　　In summary, the c-fos expression in baroreflex pathway in medulla oblongata is enhanced correspondingly with an increase in ISP. Carotid sinus perfusion with Ado can increase c-fos expression in medulla oblongata.

^*Correspondence to Prof. HE Rui-Rong. Phn: 86-311-6044121 ext.5566; Fax: 86-311-6048177;E-Mail: syho@sjz.col.com.cn.
^**联系作者. Phn: 86-311-6044121 ext.5566; Fax: 86-311-6048177;E-Mail: syho@sjz.col.com.cn

作者单位:陈　爽　张小雪^*　石葛明^*　何瑞荣^**　河北医科大学基础所生理室，　^*解剖室, 石家庄 050017

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Received 1998-06-17　　Revised 1998-09-14