Cytochrome Oxidase Activity of the Suprachiasmatic Nucleus and Pineal Gland in Rats with Portacaval Shunt

Experimental Neurology 173, 275–282 (2002)doi:10.1006/exnr.2001.7840, available online at http://www.idealibrary.com on

Cytochrome Oxidase Activity of the Suprachiasmatic Nucleus andPineal Gland in Rats with Portacaval Shunt

Laudino Lopez,* Hector Gonzalez-Pardo,* Jose Manuel Cimadevilla,* Marıa Cavas,†Marıa A. Aller,‡ Jaime Arias,‡ and Jorge L. Arias*

*Laboratorio de Psicobiologıa, Universidad de Oviedo, 33003 Oviedo, Spain; †Facultad de Psicologia, Universidad de Malaga,Malaga, Spain; and ‡Departamento de Cirugıa I, Facultad de Medicina, Universidad Complutense de Madrid, Madrid, Spain

Received March 5, 2001; accepted October 18, 2001

Rhythmic behavioral and biochemical changes havebeen observed in both human and animal models withhepatic insufficiency. The basis of all these alterationsis the principal endogenous pacemaker, the suprachi-asmatic nucleus. The aim of this work, therefore, is todetermine cytochrome c oxidase activity, a marker ofneuronal activity and oxidative metabolism, in thisnucleus in rats with portacaval shunt. In order to dothis, this enzyme was histochemically marked andquantified by computer-assisted optical densitometry.Results show a reduced cytochrome oxidase activityin the suprachiasmatic nucleus in animals with porta-caval shunts and, inversely, an increase in oxidativemetabolism in the pineal gland, another circadianstructure. However, the activity measured in a noncir-cadian brain structure, the hippocampus, which servedas a control, showed no changes with surgery. Addition-ally, locomotor activity was assessed by actimeters andrevealed a clearly reduced activity in animals with porta-caval shunt. We conclude that the suprachiasmatic nu-cleus is possibly involved in the rhythmic changes associ-ated with hepatic insufficiency. © 2002 Elsevier Science (USA)

Key Words: cytochrome oxidase; hepatic encephalop-athy; portacaval shunt; pineal gland; suprachiasmaticnucleus; locomotor activity; oxidative metabolism.

INTRODUCTION

Hepatic encephalopathy is a group of symptoms thatmay occur when there is damage to the brain andnervous system as a complication of liver disorders,like cirrhosis. This syndrome is characterized by vari-ous neurological symptoms including changes in re-flexes, consciousness and behavior that can range frommild to severe (8, 41). Since hepatic encephalopathyhas not been attributed to any one toxic substance oraltered mechanism, it is considered to result from thecombined effect of several factors such as modificationof different neurotransmitter systems, the integrityand/or function of the brain–blood barrier, and cere-

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bral energy metabolism (2, 26). One of the main symp-toms of the neuropsychiatric syndrome of hepatic en-cephalopathy is an altered sleep pattern (15, 27). Also,in cirrhosis, regardless of the etiology, circadianrhythms of several biological systems have been re-ported to be modified (5, 7, 71).

Experimentally, several models have been used toproduce hepatic insufficiency and its development intohepatic encephalopathy (8). In all of these, a change inspontaneous locomotor activity was observed, a behav-ior characteristic of circadian rhythms. Also, decreasedlocomotor activity and even changes in circadianrhythms have been observed in models of acute hepaticinsufficiency and in portosystemic encephalopathy (16,30, 78, 79).

In addition, changes in plasma melatonin levels, thehormone produced by the pineal gland (PG), have beenobserved. Production of this hormone has a clear cir-cadian rhythm (28, 46), which is dependent on envi-ronmental light conditions, with increased release atnight and less during the day. However, rats kept inconstant darkness also have a rhythmic melatonin pro-duction, but this does not occur in animals kept inconstant light (60). These results seem to suggest that,in addition to regulation by environmental light, thereis an internal pattern regulated by an endogenouspacemaker. In fact, in the rat the PG alone is neitherrhythmical nor photosensitive but depends on the pat-tern of noradrenergic stimulation of the superior cer-vical ganglia for melatonin synthesis (49). This rhythmis produced in the SCN and is relayed to the pinealgland.

Taken together, these observations suggest that he-patic conditions and changes in the rhythmic se-quences of certain biological and behavioral patternsare related. Also, as previously shown by several re-searchers, the suprachiasmatic nucleus (SCN) hasbeen found to play an important role in these rhythmicchanges. In one work, Stephan and Zucker (66) showedhow lesions of the SCN destroyed circadian rhythms offluid ingestion and locomotor activity. Tessonneaud

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276 LOPEZ ET AL.

and Locatelli (69) showed that stimulation of the PG bythe superior cervical ganglion is dependent on the SCNvia its connection with the paraventricular nucleus.Moreover, other authors have reported that SCN le-sions modify the production of adrenal corticosteroidsand the rhythmic production of ACTH (11, 48).

Since there appears to be an association betweenhepatic pathology and certain rhythmic changes, ourwork was aimed at observing changes in the oxidativemetabolism of two structures which show a clear en-dogenous rhythm: the SCN and the PG. In order to dothis we used a well-known histochemical marker ofnervous system activity (75): the mitochondrial en-zyme cytochrome c oxidase (CO; EC 1.9.3.1), the activ-ity of which is related to neuronal energetic metabo-lism. Previous studies have shown changes in energyconsumption in the SCN measured by other techniqueswidely used for determining energetic metabolism suchas the baseline incorporation of 2-[14C]deoxyglucose(62), cyclic AMP consumption (53), and CO activity (44,52). However, this is the first time that the quantita-tive histochemical technique for CO has been used toassess the changes caused by hepatic insufficiency.

MATERIALS AND METHODS

Animals and Surgery

Twenty-four Wistar rats from the vivarium of OviedoUniversity, Oviedo, Spain, were used. The meanweight of rats at the start of the experiments was289 6 34 g. All the animals had ad libitum access tofood (Rat A040 maintenance diet, Panlab, Barcelona,Spain) and tap water and were maintained at a con-stant room temperature (20 6 2°C), with a relativehumidity of 65 6 10% and an artificial light–dark cycleof 12 h (0800–2000 h/2000–800 h). Rats were housedtogether in groups of four in polycarbonate cages (42 365 3 15 cm, Letica, Barcelona, Spain). All the experi-ments were performed according to the European Com-munities Council Directive of November 24, 1986.

The portacaval shunt (PCS) operation was per-formed (43) with a slight modification consisting of thetotal clamping of the inferior vena cava during PCSperformance (4). The time in which the portal vein andinferior vena cava were clamped for anastomosis didnot exceed 15 min. In the SHAM group, after a bilat-eral subcostal laparotomy with prolongation to the xi-phoid apophysis was performed, the vena porta and thecava inferior were clamped for 15 min (8). All surgicaloperations were performed on animals anesthetized byinhalation of ether. The postsurgical recovery periodwas 60 days.

Assessment of Locomotor Activity

The environmental light conditions were the samethroughout the experiment as a 12-h light–dark cycle

(lights on from 0800 to 2000 h). This room was keptclosed and sound-proofed during the experiment. Ani-mals were given ad libitum access to food and water atall times. There were a total of 12 animals in eachgroup (PCS and SHAM).

Locomotor activity was assessed using infrared sen-sory units, which permitted quantification of the ani-mal’s movements (Actisystem, Panlab). Each animalwas placed in its cage on a sensory unit connected to adevice for recording movement, which was, in turn,connected to a personal computer. A computer programwas used to record the activity of each animal over apreviously determined time period. Recordings in eachgroup were made at hourly intervals over a 3-day pe-riod. In all cases, animals were placed on the sensoryunits 4 days before their movements were recorded(conditioning time).

CO Histochemical Labeling of Nervous Tissue

The animals were anesthetized by inhalation of ethylether and were vascularly perfused with 0.1 M phos-phate buffer (pH 7.6). All of the animals were sacrificedbetween 1200 and 1300 h (lights-on period). The brainswere sectioned on the coronal plane, including the opticchiasm region, according to Paxinos and Watson’s atlas(58), and coated with a cryoprotective gel (OCT com-pound, Miles Inc., Redding, CA, USA), frozen withfreon-22 cooled by liquid nitrogen, and stored at 270°C.They were sectioned in a cryostat (Jung-Reichert,Frigocut-250, Heidelberg, Germany) at 220°C, and 20-mm-thick sections were obtained. The sections werehistochemically processed to reveal CO by diaminoben-zidine according to a modification of the Wong-Rileymethod (75) by Sukekawa (68). Alternate sections wereNissl stained (with cresyl violet), in order to locate theSCN (Fig. 1). The same procedure was applied to locatethe pineal gland and hippocampus.

To quantify CO activity, the method described byGonzalez-Lima and Cada (33) was used. Our researchgroup has previously used an adaptation of this tech-nique in developmental brain studies (34). Briefly, ali-quots from entire rat brain homogenates were quanti-fied by the spectrophotometrical method described byWharton and Tzagoloff (73). These aliquots containeddifferent mixtures of fresh and heat-inactivated brainhomogenate. Cryostat sections from these homogenateswere histochemically stained for CO together with theother brain sections in the same staining bath. Using thisprocedure a calibration curve was obtained between realCO activity measured previously in the aliquots frombrain homogenates in specific CO units (1 specificunit 5 1 mmol cytochrome c oxidized/min and gramtissue (w/w) at pH 7 and 23°C) and the densitometryof the histochemical CO stain was measured by acomputer image analyzer attached to a microscope(Leica, Q-Win, Wetzlar, Germany). With the help of

277CYTOCHROME OXIDASE ACTIVITY IN RATS WITH PORTACAVAL SHUNT

this conversion curve, the densitometric values ob-tained with the image analyzer from the suprachias-matic nucleus were transformed to CO-specific units.Brain regions were selected on the screen using a digi-tizer board as input device. The image pixels insidethese regions are automatically converted to gray tonesthat correspond to integrated mean optical density val-ues. The microscope light source remains constantacross measures, since its intensity is computer con-trolled.

Nine of 12 animals from each experimental group(PCS and SHAM) were used for CO histochemistry.For each animal, six measurements were obtained inthe suprachiasmatic nucleus (n 5 9 each group). Addi-tionally, three measurements were taken in the pinealgland and seven measurements were collected from thehippocampus (CA1 and CA3 areas) per animal in eachexperimental group (n 5 7 each group). Fewer animalswere used to evaluate CO activity in both the pinealgland and the hippocampus, due to the high level ofhomogeneity in CO activity shown by these brain re-gions.

Statistical Analysis

Differences in organ and body weights from bothgroups were analyzed using Student’s t tests. The nor-mal distribution of the CO measures was first evalu-ated using a Kolmogorov–Smirnov test with Lillefor’scorrection. Then, a repeated-measures two-way analy-sis of variance (ANOVA) followed by Tukey’s test (withSpjøtvoll-Stoline correction for unequal sample sizes)for multiple comparisons was applied to CO data (thebrain region selected was the repeated-measures factor

FIG. 1. Photomicrographs of coronal sections taken from ratsCytochrome c oxidase histochemistry. Please note the anatomical coof enzyme activity of this nucleus.

and surgical treatment group was the independentfactor).

Locomotor activity data from PCS and SHAM ratswere compared using Student’s t test for independentmeasures and its nonparametric equivalent (Mann–Whitney’s U test) when normality (Kolmogorov–Smir-nov) and equal variance (Levene median test) testswere not passed.

RESULTS

Testing the Portacaval Shunt

Results revealed that PCS animals showed a mini-mal increase in body weight 60 days after intervention(14% of their initial weight compared to a mean in-crease of 45% in the SHAM group; Student’s t test; P ,0.001; see Table 1). The liver weight in PCS animals

the level of the suprachiasmatic nucleus. (A) Nissl staining. (B)ation of the limits from both staining techniques and the high level

TABLE 1

Body Weight, Hepatic Weight, and Testicular Weightof Male Rats with Portacaval Shunts

GroupInitial body

weightFinal body

weightHepaticweight

Testesweight

SHAM(n 5 12) 281 6 38 407 6 29 13.32 6 1.02 3.62 6 0.24

PCS(n 5 12) 296 6 21 337 6 45* 7.85 6 1.53* 1.89 6 0.51*

Note. PCS, portacaval shunted group. SHAM, animals with lapa-rotomy and 15 min of vena porta and inferior cava being clamped.Final, hepatic, and testes weights were measured 60 days aftersurgery. Mean 6 SD; *P , 0.05 vs SHAM.

atrrel

278 LOPEZ ET AL.

had a mean decrease of 41% versus SHAM group (P ,0.001; Table 1). Testicular weight decreased in the PCSanimals by 47% compared to animals in the SHAMoperated group (P , 0.001; Table 1). Finally, no post-mortem abnormal changes were observed in the anas-tomosis in any of the operated animals.

CO Activity in Suprachiasmatic Nucleus,Pineal Gland, and Hippocampus

The photomicrographs (Fig. 1) indicate CO histo-chemistry in the SCN of sham rats. Statistical analysisof CO data by ANOVA showed a highly significantinteraction between surgical treatment and brain re-gions selected (F(3,36) 5 11.22; P , 0.0001). Posthocanalysis of the interaction showed significantly lowerCO values in the SCN from the PCS group (Tukey’stest; P , 0.05) and higher values in the PG (Tukey’stest; P , 0.01) (Fig. 2). In the hippocampus, posthocanalyses showed no statistically significant differencesbetween treatments (PCS vs SHAM) in both the CA1and the CA3 areas (Fig. 2).

FIG. 3. Average locomotor activity of each group (mean 6 SEM,n 5 12 per group) during the light and dark periods and over theentire 24-h period (total activity); *P , 0.0001 (Student’s t test). PCS,portacaval shunted group; SHAM, sham-operated group.

FIG. 2. Determination of cytochrome oxidase (CO) activity(mean 6 SD) in the suprachiasmatic nucleus (SCN, n 5 9 per group),the pineal gland (PG, n 5 7 per group), and hippocampal regions(CA1 and CA3, n 5 7 per group). PCS, group with portacaval shunt;SHAM, sham-operated group; *P , 0.05, **P , 0.01 (Tukey’s test).

Locomotor Activity

The statistical analysis applied to the total locomotoractivity results revealed significant differences be-tween the PCS and the SHAM groups (Student’st(22) 5 2.66, P , 0.05, Figs. 3 and 4). However, therewere no differences between both groups during thelight phase (Mann-Whitney’s U test, P . 0.9), but asignificant result was obtained during the dark phase(Mann–Whitney’s U test, P , 0.01).

DISCUSSION

The changes in CO activity found in the presentstudy between the PCS and the SHAM groups (Fig. 2)could be explained by two hypotheses: (i) a shift inthe periods of CO activity caused by PCS and (ii) anadditional consequence of general morpho-functionalchanges in the nervous system after PCS that involveendogenous circadian rhythms.

FIG. 4. Plot of the mean activity per hour (mean 6 SEM) of eachgroup over the entire 24-h period, across 1-h intervals. The increasedactivity in the dark phase of both groups can be observed with asmaller amplitude in the portacaval shunted group and no differ-ences between groups during the light period. The “MESOR” lineindicates the mean basal activity during the entire period. PCS,portacaval shunted group; SHAM, sham-operated group.

279CYTOCHROME OXIDASE ACTIVITY IN RATS WITH PORTACAVAL SHUNT

In relation to our first hypothesis, the SCN producesintrinsic circadian rhythms of biochemical and physi-ological parameters, such as glucose uptake (62, 74),spontaneous electrical activity (9), and ATP levels (77).In this way, our group has recently reported that theCO activity of the SCN changes according to lightconditions (44). These results suggest that the differ-ences in CO activity in both the SCN and the PG couldreveal a displacement in CO activity.

As for our second hypothesis, the changes observedin CO activity could be a consequence of other alter-ations produced by PCS. However, our results differfrom several works dealing with metabolic activity af-ter PCS (24). A possible explanation for the discrepan-cies found in the results would be that most of the otherstudies are based on techniques which measure ratesof cerebral metabolic glucose utilization (CMRGlc) (22).This could explain why the results reported in severalworks differ (24), since these authors found differentresults in CMRGlc after PCS when deoxy-[14C]glucose or6-[14C]glucose was used. Differences in the hippocam-pal metabolism of PCS rats have also been found. Twofactors could account for these discrepancies (i) COactivity is only indirectly related to CMRGlc or (ii) COhistochemistry has a better anatomical resolution thanthe above-mentioned autoradiographic methods andcan clearly distinguish the anatomical subdivisions ofthe hippocampus (CA1, CA3). The present data confirmthose reported by Cruz and Dienel (22) with the deoxy-[14C]glucose technique regarding the absence ofchanges in the hippocampus after PCS.

From our results in the SCN, we can see that thisbrain region is anatomically well defined with CO his-tochemistry (Fig. 1). This explains why other authorshave obtained similar results with this or other quan-titative methods for CO or CMRGlc (33, 36, 39, 52).However, no differences in the CMRGlc of the SCN havebeen found in studies using another marker (2-[14C]deoxyglucose) (32). Again, these results must beinterpreted with caution, not only because of the dif-ferences in the techniques used to measure CMRGlc, butalso because of the differences between CMRGlc andoxidative metabolism (CO). However, these techniquesare related since they can both be considered as indi-rect indices of energy metabolism, i.e., glucose metab-olism in the central nervous system (32). Additionally,there is a clear correlation between energy (stored inform of ATP) and CO levels (38). Moreover, CO activityhas already been used as a marker of oxidative metab-olism and neuronal activity in neurodegenerative pro-cesses (Alzheimer’s disease) (13).

On the other hand, the changes found in oxidativemetabolism in both the SCN and the PG could belinked to impairments developed by PCS animals insome neurotransmitter systems (10). Both histamineand serotonin have been shown to increase in rats withPCS (26, 29, 70). Histamine could be involved in regu-

lation of the neuroendocrine system, the sleep–wakecycle, feeding, and locomotor activity via connectionsbetween the SCN and the tuberomamillary nucleus(55, 56, 76). Moreover, there is a clear relationshipbetween serotonin and the SCN since one of the mainafferents to the SCN comes from the raphe nuclei,which represent around 12% of the nerve terminals ofthe SCN (67). In agreement with our results of alteredCO activity in PCS rats, in vitro studies have shownthat histamine appears to act on the SCN itself, pro-ducing phase changes on a neuronal level (19), andthat serotonin acts on glucose uptake (23). Therefore,the increase in histamine and serotonin levels ob-served in PCS animals could be in part responsible foran impaired oxidative metabolism (45).

Our results also suggest an important role for astro-cytes in SCN activity (50, 59) and alterations in theglial cell population from PCS rats are frequently re-ported (1, 54). However, although glial cells have anegligible CO activity (their metabolism is basedmainly on anaerobic glycolisis (75)), their close rela-tionship with neurons would explain a possible indirectalteration of neuronal CO activity. Moreover, changesin gonadal hormones in PCS rats (37, 72) influenceSCN activity since they have activational properties onseveral aspects of circadian rhythms (40, 51, 57).

In relation to the CO activity in the PG, rats alsohave altered oxidative metabolism in the PG, withhigher values of CO activity between 12 and 13 h of thelight phase (Fig. 2). Zee et al. (79) found higher diurnalplasma melatonin levels in PCS animals than inSHAM animals, possibly related to the increased COactivity observed in the present study. However, theelevated melatonin levels could be caused by an im-paired hepatic catabolism in PCS rats (64). There areseveral possible explanations for this increase. It couldcaused by alterations in the SCN itself (69) or in itshistaminergic afferents (47), by increased tryptophanlevels, or by changes in the pineal glial cells involved inthe glutamate and GABA metabolism via glutaminesynthetase (42). On the other hand, the PG is also atarget organ for estradiol and testosterone, which canhave greatly altered plasma levels in rats with PCS(35, 37, 72).

The PG plays a role in maintaining circadianrhythms because of its ability to modulate the SCNitself (12, 25, 31). Moreover, the diurnal increase inmelatonin could contribute to a decrease in SCN oxi-dative metabolism, since, in short experiments on theSCN, melatonin reduces the high level of metabolicactivity found during the light phase (61).

Regarding the spontaneous locomotor activity of an-imals with PCS, a clear circadian rhythm is observed60 days after surgery, with a clear increase in activityduring the dark or active phase. This change is knownto be regulated by the light–dark cycle since the activ-ity curve is dependent on the presence or absence of

280 LOPEZ ET AL.

environmental light. In this way, total locomotor activ-ity during the complete cycle (24 h) is significantlylower in this group compared to SHAM animals (Fig.3). In general, hypoactivity of animals with PCS, aswell as in other experimental models of hepatic en-cephalopathy, is a common finding in studies that useeither the open-field or actimeters (29, 63, 78).

This clear hypoactivity observed in rats with PCScannot be directly due to the increase in ammoniaresulting from the portacaval shunt since administra-tion of ammonium acetate in control rats does notinduce this behavior (6). Also, the use of neomycinimproves impaired activity without decreasing ammo-nia levels (17). It is more likely to be caused by in-creased serotonin levels (5) or by abnormal functioningof the SCN itself (21). However, our data differ fromthose published by other authors which report in-creased activity during the light phase with a decreasein total activity and even a loss of circadian rhythms in50% of the animals (20, 65, 79). In the present study,animals with PCS had similar values to SHAM ani-mals during the light period (Fig. 4). These data con-firm those reported by other authors, which found adecreased locomotor activity during the dark phase (3,63). The latter decrease of locomotor activity duringthe dark phase cannot be attributed to deficits in themotor system, since the values during the light phaseare identical to those from the control group.

In conclusion, our results show, for the first time,changes in CO activity in the SCN and PG in rats withPCS. Moreover, reduced locomotor activity was ob-served in rats with PCS. Our findings agree with datafrom human patients with cirrhosis and hepatic en-cephalopathy, in relation to an abnormal circadiantime-keeping system: elevated melatonin levels duringdaytime hours, sleep disorders, or changes in severalbiochemical parameters in blood (8, 14, 18, 64).

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