Acta Physiologica Sinica

Dec. 2001, 53 (6), 445¡«450

Renal ischemiaª²induced Fos expression in catecholaminergic neurons of rats

DING Yanª²Feng1, ZHANG Xiaoª²Xue2, WANG Yiª²He3, SHI Geª²Ming2, HE Ruiª²Rong1,*

(~1Departments of Physiology and ~£²Anatomy, Hebei Medical University, Shijiazhuang 050017; £³Department of Physiology, Wujing Medical College, Tianjin 300162)

Abstract: The present study was undertaken to define whether the renal artery occlusion (RAO) would activate the catecholaminergic neurons in the brainstem nuclei by double immunohistochemical method for detecting Fos and tyrosine hydroxylase. The results are as follows: (1) while the basal expression of Fos was relatively low in the brainstem, RAO was capable of inducing a robust Fosª²like immunoreactive neurons in the nucleus tractus solitarius (NTS), area postrema (AP), nucleus paragigantocellularis lateralis (PGL) and locus coeruleus (LC); (2) numerous catecholaminergic neurons in NTS, AP, PGL and LC could be activated by RAO as shown by Fos expression; and (3) these responses to RAO were significantly attenuated by pretreatment with an adenosine receptor antagonist 8ª²phenyltheophylline. The results suggest that RAO can activate a large number of neurons including some catecholaminergic neurons in the brainstem nuclei. Such effects of renal ischemia may be attributed to RAOª²induced adenosine release from the kidney which subsequently activates renal afferents.

Key words: renal ischemia; brainstem; Fos immunohistochemistry; tyrosine hydroxylase; adenosine; 8ª²phenyltheophylline

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ÕªÒª: ʵÑéÓ¦ÓÃFosµ°°×ºÍÀÒ°±ËáôÇ»¯Ã¸(tyrosine hydroxylase, TH)µÄË«ÖØÃâÒß×黯·½·¨, ¹Û²ìÉöÔදÂö×è¶Ï(renal artery occlusion, RAO)ÊÇ·ñ¼¤»îÄÔ¸ÉÖкËÍŵĶù²è·Ó°·ÄÜÉñ¾Ôª¡£ËùµÃ½á¹ûÈçÏÂ: (1) ÄÔ¸ÉÖÐFosÑùµ°°×µÄ»ù´¡ÐÔ±í´ïµÍ; RAO¿ÉÓÕ·¢¹ÂÊøºË(nucleus tractus solitarius, NTS)¡¢ ×îºóÇø(area postrema, AP)¡¢ ¾Þϸ°ûÅÔÍâ²àºË(paragigantocellularis lateralis, PGL)ºÍÀ¶°ß(locus coeruleus, LC)ºËÍÅÖÐÐí¶àÉñ¾ÔªÏÔʾFosÑùÃâÒß·´Ó¦(Fosª²like immunoreactivity, FLI)¡£(2) NTS¡¢ AP¡¢ PGLºÍLCºËÍÅÖк¬Óн϶àµÄ¶ù²è·Ó°·ÄÜÉñ¾Ôª; RAOÄܼ¤»îÆäÖеIJ¿·Ö¶ù²è·Ó°·ÄÜÉñ¾Ôª¡£(3)ÏÙÜÕÊÜÌå×è¶Ï¼Á8ª²±½²è¼î¿ÉÃ÷ÏÔ¼õÈõRAOËùÖµÄÉÏÊöЧӦ¡£ÒÔÉϽá¹û±íÃ÷, ÉöÔà¶ÌÔÝȱѪÄܼ¤»îÄÔ¸ÉÄÚµÄһЩÉñ¾ºËÍÅÒÔ¼°ÆäÖеIJ¿·Ö¶ù²è·Ó°·ÄÜÉñ¾Ôª¡£´ËЧӦ¿ÉÄÜÊÇÉöȱѪʱÏÙÜÕÊÍ·Å×÷ÓÃÓÚÉöÄÚÏÙÜÕÊÜÌåºóÒýÆðÉö´«ÈëÉñ¾»î¶¯Ôö¼ÓµÄ½á¹û¡£

¹Ø¼ü´Ê: ÉöȱѪ; ÄÔ¸É; FosÃâÒß×éÖ¯»¯Ñ§·´Ó¦; ÀÒ°±ËáôÇ»¯Ã¸; ÏÙÜÕ; 8ª²±½²è¼î

ѧ¿Æ·ÖÀàºÅ: Q463; R331.1

A large body of evidence has shown that the afferent information from renal receptors influences the activity of neurons at different levels of the neuraxis, including the brainstem nuclei£Û1¡«5£Ý. A direct projection from the kidney to the brainstem has been found£Û1,2£Ý. Neurons within the brainstem nuclei respond to the stimulation of renal nerves and receptors£Û3¡«5£Ý. Our previous studies have shown that renal transient ischemia induces an increase in electrical activity of renal afferents and neurons in rostral ventrolateral medulla (RVLM), and enhances Fos expression in RVLM neurons£Û6,7£Ý. These findings imply that renal ischemia may activate the brainstem nuclei. Moreover, it is known that brainstem nuclei such as the nucleus tractus solitarius (NTS), paragigantocellularis lateralis (PGL), area postrema (AP), locus coeruleus (LC) etc. contain catecholaminergic cell groups£Û8£Ý.

¡¡It is well established that Fos protein expression is a metabolic marker of neuronal activation£Û4,9¡«12£Ý, and that the catecholamineª²synthesizing enzyme tyrosine hydroxylase (TH) is a symbol of catecholaminergic neurons. The present study was undertaken to determine whether renal ischemia could induce Fos expression change associated with the activation of the catecholamineª²containing neurons in brainstem nuclei by double immunohistochemical techniques.

1MATERIALS AND METHODS

1.1 AnimalsMale Spragueª²Dawley rats weighing 250¡«300 g were housed in colony cages under conditions of controlled illumination (12/12 h light/dark cycle), humidity and temperature (22¡À2¡æ) for at least 3 days prior to the surgical procedure. Food and tap water were provided ad libitum. All rats were anesthetized with urethane (1 g/kg, ip), and moved into the nearby laboratory to avoid the environmental stimuli.

1.2 Drugs and reagents8ª²phenyltheophylline (8ª²PT) and TH monoclonal antiserum were purchased from Sigma Chemical Co. The other immunohistochemical reagents were the products of Santa Co. 8ª²PT was dissolved in normal saline.

1.3 General preparationsRats breathed spontaneously through an intratracheal tube. A polyethylene catheter was inserted into the femoral vein for drug administration. Bilateral buffer nerves were sectioned and the completeness of barodenervation (sinoaortic denervation) was verified by the method described in detail elsewhere£Û7,11£Ý. The left renal artery was dissected free via a retroperitoneal approach in all animals£Û5¡«7£Ý. A 3¡«0 silk thread was passed around the renal artery near its junction to the aorta, and the ends of suture were threaded through a segment of polyethylene tube to form a snare, which was used to occlude the artery as required. The body temperature of animals was maintained at 37¡«38¡æ.

1.4 Experimental protocolsA period of 30 min was allowed for stabilization after the operation. The experimental animals were divided into the following groups. Group 1 (n=5): rats were only subjected to sham operation. Group 2 (n=5): rats underwent 10ª²min renal artery occlusion (RAO). Group 3 (n=5): 8ª²PT (10¡¡mg/kg) were given via femoral vein. Group 4 (n=5): before RAO, 8ª²PT (10 mg/kg) was administrated. After 2 h, animals were deeply anesthetized with urethane and perfused through the ascending aorta with 100 ml of normal saline, followed by 400 ml iceª²cold 4% paraformaldehyde and 0.2% picric acid in 0.1¡¡mol/L, pH 7.5 phosphateª²buffered saline (PBS). The brains were postª²fixed in the same fixative for 4 h and subsequently cryoprotected in 30% sucrose overnight at 4¡æ. Coronal sections (40 ¦Ìm) of the brain were cut in a cryostat. Sections of the brainstem were collected in PBS for further processing. Experimental rats and their controls were processed on the same day and the brain sections were reacted together to avoid the variability due to the staining procedures.

1.5 Double immunohistochemical labeling procedure£Û11£ÝSections were incubated overnight at 4¡æ with rabbit antiª²cª²fos antiserum (1¡Ã500) in 0.3% Triton Xª²100/PBS. In the following day, sections were incubated with biotinylated antiª²rabbit IgG (1¡Ã300) for 4 h and then with avidinª²biotinª²peroxidase system (1¡Ã300) for 1 h at room temperature. After a rinse in PBS, the reaction product was visualized by incubating sections in hydrogen peroxidase and diaminobenzidine tetrahydrochloride (DAB) with 0.01% NiCl2¡¤H2O for intensification. Sections were then incubated overnight in a mouse monoclonal antiserum raised against TH (1¡Ã8 000) and biotinylated horse antiª²mouse IgG (1¡Ã200) followed by the ABC method and DAB/H2O2 to visualize the reaction product under a light microscopy. Sections were mounted on chrome alum gelatinª²coated slides, dehydrated in alcohol and xylene, and coverslipped.

1.6 Cell count and data analysisTissue sections were examined under a standard light microscope. The labeled cell number per section was counted in each region defined by the brain altas of Paxinos and Watson for each animal£Û13£Ý. All data were expressed as means¡ÀSE. The differences of the number of neurons positive for TH activity, Fos expression or both were examined by analysis of variance (ANOVA) followed by the unpaired t test for betweenª²group comparisons. Statistical significance was accepted when P<0.05.

2RESULTS

¡¡Fosª²like immunoreactive (FLI) neurons were expressed as blackª²stained round or oval bodies representing neuronal nuclei, whereas TH immunoreactivity was visualized as a brown color within perikarya, dendrites and axons. The doubleª²labeled neurons were visualized as brownª²stained perikarya containing a clearly visible black nucleus.

2.1 Fos expression

In rats with sham operation, few neurons showed FLI throughout the brainstem. RAO led to a robust Fos expression in NTS, AP, PGL and LC except raphe nuclei and periaqueductal gray. Pretreatment with 8ª²PT significantly reduced the RAOª²induced Fos expression (Figs.1 and 2).

Fig.1A¡«H

Fig.1.Photomicrographs illustrating the Fos immunoreactive (FLI) neurons (clear arrowheads), tyrosine hydroxylase (TH) labeled neurons (filled arrowheads) and doubleª²labeled neurons (arrows) in nucleus tractus solitarius (NTS, A¡«D), area postrema (AP, E¡«H), nucleus paragigantocellularis lateralis (PGL, I¡«L) and locus coeruleus (LC, M¡«P) in the rats with sham operation¡¡(A, E, T and M), renal artery occlusion¡¡(RAO, B, F, J and N), 8ª²phenyltheophylline (8ª²PT, C, G, K and O) and 8ª²PT+RAO¡¡(D, H, L and P).

Fig.2.Histograms illustrating the counts of FLI neurons in NTS, AP, PGL and LC of the brainstem.~**P<0.01, ~***P<0.001 compared with the sham operation group; ~++P<0.05 compared with RAO group.

Fig.3.Histograms illustrating the counts of doubleª²labeled neurons for FLI and TH in NTS, AP, PGL and LC of the brainstem. ~*P<0.05, ~**P<0.01, ~***P<0.001 compared with the sham operation group;¡¡~++P<0.05 compared with RAO group.

2.2 TH immunoreactivity

¡¡In sham operation animals, numerous TH immunoreactive neurons were observed in NTS, AP, PGL and LC. Following RAO or intravenouse injection of 8ª²PT, the number of TH immunoreactive neurons showed no change (Fig.1). Few TH immunoreactive neurons were seen in raphe nuclei and periaqueductal gray.

2.3 Coª²localization of Fos and TH immunoreactivity

¡¡A few doubleª²labeled neurons were found in NTS, AP, PGL and LC of rats with sham operation. RAO led to a significant increase in the number of doubleª²labeled neurons per section of these nuclei. The doubleª²labeled neurons expressed during RAO were significantly reduced by pretreatment with 8ª²PT (Figs.1 and 3).

3DISCUSSION

¡¡In the present study, it is demonstrated that RAO induces Fos protein expression in the medulla oblongata. Although the basal expression of Fos is relatively low in the central nervous system£Û10,12£Ý, Fos protein transiently expressed in neurons after a variety of stimulation has been used as a metabolic marker in neuronal tracing£Û9£Ý. A subpopulation of TH immunoreactive neurons in the NTS, AP, PGL and LC was also found to express Fos protein following RAO. It has been well established that electrical stimulation of renal afferent nerves induces an increase in the number of FLI neurons within the supraspinal sites£Û4£Ý. The electrical activity of brainstem nuclei might be affected by occlusion of the ureter or renal vein, and electrical stimulation of renal afferent nerves£Û3,5,7£Ý. In addition, the neurons within brainstem nuclei may be a link in the baroceptor reflex circuity£Û12£Ý. In our present experiments, the rats with sinoaortic denervation were used to exclude the indirect effect from baroreflex on brainstem neurons.

¡¡It is well known that there are chemoreceptors and mechanoreceptors in the kidney. As renal artery is occluded, the ischemic kidney may release some active substances (adenosine, etc). These substances released from ischemic kidney are capable of activating the chemoreceptors (R1 and R2)£Û14¡«16£Ý. Vizzard et al. point out that mechanoreceptors are also activated by RAO£Û5£Ý. Activation of renal receptors alters the activity of renal afferent and spinal neurons. The information is then conveyed to a variety of supraspinal sites including the brainstem£Û3¡«7, 14¡«16£Ý. Moreover, renal afferents may also project directly to brainstem neurons£Û2£Ý. In our experiments, the FLI neurons as well as the doubleª²labeled neurons induced by RAO were significantly decreased by pretreatment with an adenosine receptor antagonist 8ª²PT, indicating that adenosine was released in the ischemic kidney and resulted in activation of adenosine receptors to stimulate the renal chemore~ceptors and mechano receptors. In previous studies, we documented that RAO could increase the renal afferent activity, electrical activity and Fos expression of the PGL neurons. Such effects induced by RAO could be partially abolished by 8ª²PT£Û6,7£Ý. It is thus conceivable that, in addition to adenosine, some other active substances might be released in ischemic kidney and involved in the activation of renal receptors. The precise nature of other active substances remained to be clarified.

¡¡On the basis of our result about TH immunoreactivity, PGL, NTS, AP and LC contain a population of catecholaminergic neurons. This result is consistent with that in our previous experiments£Û11£Ý. The number of doubleª²labeled neurons was increased after RAO, indicating that some catecholaminergic neurons were activated by RAO. It is well known that catecholaminergic neurons in the brainstem nuclei may be involved in the modulation of sympathetic activity, and that catecholamines are presumptive neurotransmitters or neuromodulators involved in the reflex control of circulation and release of hypothalamic neurohormones or peripheral catecholamines£Û11,17,18£Ý. However, the catecholaminergic cell groups were divided into noradrenergic cell groups, adrenergic cell groups and dopaminergic cell groups£Û19,20£Ý. So the subtypes of RAOª²induced catecholaminergic neurons need to be elucidated.

¡¡In summary, RAO results in a marked increase in FLI and Fosª²TH doubleª²labeled neurons in PGL, AP, NTS and LC. Adenosine released in the ischemic kidney may be involved in such effects.

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Received 2001ª²03ª²03Accepted 2001ª²04ª²11

This study was supported by the National Natural Science Foundation of China (No.30070282).

ª³Corresponding author. Tel: 86ª²311ª²6062490; Fax: 86ª²311ª²6062490; Eª²mail: syho@hebmu.edu.cn