Decreased sympathetic innervation of spleen in aged fischer 344 rats

Neurobiology of Aging, Vol. 8, pp. 15%165. ©PergamonJournals Ltd., 1987. Printedin the U.S.A.

019%4580/87$3.00 + .00

Decreased Sympathetic Innervation of Spleen in Aged Fischer 344 Rats S U Z A N N E Y. F E L T E N , 2 D E N I S E L. B E L L I N G E R , T I M O T H Y J. C O L L I E R , P A U L D. C O L E M A N A N D D A V I D L. F E L T E N

D e p a r t m e n t o f Neurobiology and A n a t o m y , University o f R o c h e s t e r School o f Medicine an-d Dentistry Rochester, N Y 14642 R e c e i v e d 1 A u g u s t 1986 FELTEN, S. Y., D. L. BELLINGER, T. J. COLLIER, P. D. COLEMAN AND D. L. FELTEN. Decreased sympathetic innervation of spleen in aged Fischer 344 rats. NEUROBIOL AGING 8(2) 15%165, 1987.--Splenic noradrenergic innervation in young adult and aged Fischer 344 rats was examined using fluorescence histochemistry for catecholamines and high performance liquid chromatography with electrochemical detection (LCEC) for the quantitation of norepinephrine (NE). In young adult rats, abundant noradrenergic plexuses followed the vasculature and trabeculae into splenic white pu~p. In aged rats, noradrenergic innervation was reduced in density and in overall intensity of fluorescence, and splenic NE levels were significantly lower. The relationship between diminished noradrenergic innervation and diminished immune responsiveness in aging mammals, while not clear on a causal level, is presented as a hypothesis for further testing. Noradrenergic innervation Catecholamines

Spleen

Aging

Histofluorescence

LCEC

Immune system

the sensitivity to catecholamines in vitro is diminished, in Fischer 344 rats [39]. Lymphoid organs also may show agerelated noradrenergic depletion. Preliminary reports from our laboratories have noted an age-related diminution of NE in popliteal and mesenteric lymph nodes in various strains of mice, including New Zealand black mice (NZB), a genetic strain associated with autoimmune hemolytic anemia, and NZB x NZW F~ hybrid mice, a strain associated with lupus-like syndrome [2,53]. Capocelli et al. [11] have presented preliminary quantitative data on catecholamine innervation of the spleen in aging C57BL/6 mice showing stability of fiber sensity up to 30 months of age followed by sharp decline. The present study examined 8 month old (young adult) and 27 month old (aged) male Fischer 344 rats, and further subdivided the aged rats based upon their response to novel gustatory stimuli (gustatory neophobia), a behavioral task that has been shown to be sensitive to brain NE depletion resulting from dorsal NE bundle lesions in young rats [44] or as a consequence of aging [12]. Thus, it was of interest to determine whether age-related declines in central NE were predictive of changes in peripheral NE.

THE autonomic nervous system, through its extensive innervation of both primary and secondary lymphoid organs [8, 9, 14-16, 29, 50, 51], may be a prime efferent system for communication between the nervous and immune systems. Noradrenergic fibers distribute around the vasculature and within the parenchyma of lymphoid organs; in secondary lymphoid organs such as spleen, lymph nodes, and gutassociated lymphoid tissue, these fibers distribute to areas which contain primarily T lymphocytes, zones of antigen presentation, or zones where lymphocytes exit the organ [10, 14-16, 19, 45, 4%51]. Denervation of spleen or lymph nodes by surgical means or by 6-hydroxydopamine administration results in altered immune responses to antigen challenge, although the magnitude and direction of change of the antibody responses differ depending on the method of denervation, the time of denervation, and the type of immune challenge. These studies are discussed in detail by Livnat et al. [29]; in general, in animals chemically denervated with 6-hydroxydopamine (6-OHDA) as adults, antibody responses are suppressed, while in animals denervated with 6-OHDA at birth or surgically denervated as adults, antibody responses are augmented. These studies suggest complex primary and secondary interactions of NE with many regulatory systems in spleen and lymph nodes (discussed in detail by Felten et al. [16]). In some peripheral target structures, such as the heart, NE derived from sympathetic fibers is reduced with age, and

METHOD

Experimental Design Adult male Fischer 344 rats (from Charles River Labs obtained through the National Institute on Aging) were

1Supported by Grant N00014-84-K-0488 from the Office of Naval Research, 1 F32 NS07980-01 from NIMH (NINCDS), and by NIH Training Grant AG T32 107. ~Requests for reprints should be addressed to Suzanne Y. Felten, Ph.D., Department of Neurobiology and Anatomy, Box 603, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642.

159

60

FELTEN

ET AI

FIGS. 1-6.

S P L E N I C N E I N N E R V A T I O N : A N A G I N G STUDY examined at 8 and 27 months of age. All animals were tested for gustatory neophobia (see Collier et al. [12] for details of testing) and the 27 month old rats were divided into two groups, non-neophobic ( " n o r m a l " ) aged and neophobic aged groups, based upon this behavioral testing. Previous evidence suggests that these neophobic aged rats exhibit a 15-30% decline in locus coeruleus NE histofluorescence (a correlate of NE content) compared with aged " n o r m a l " rats [12]. Six animals from each group were sacrificed by decapitation, and the brains and spleens were removed for examination.

Glyoxylic Acid Method of Histofluorescence jbr Catecholamines Spleens were cut in cross section into four pieces of approximately equal size, frozen on dry ice, and stored in liquid nitrogen until examined. Cross sections of 15/xm thickness were cut from a middle piece of each spleen on a cryostat at -30°C. These sections were prepared using glyoxylic acid condensation for histofluorescence of catecholamines, modified from the SPG (sucrose-potassium phosphateglyoxylic acid) method of de la Torre [13]. Improved consistency of fluorescence was obtained by working in a laboratory with almost constant temperature and humidity, and keeping all steps identical for each sample. Sections were examined as they were prepared, and were photographed the next day on a Nikon fluorescence microscope equipped with epi-illumination accessories.

Morphometric Analysis For quantitation of NE fiber density one section from each of three animals per group was randomly selected, and all fluorescent profiles in each of these sections were photographed at the same magnification (x400) onto 35 mm slides. Slides were projected onto a 6 x 6 squares per inch grid, and the intersections of these lines with projected NE fluorescent profiles were counted. Points of intersection were totalled for each section of spleen examined, and the percentage of the volume density of N E profiles per spleen section was calculated based upon the actual magnification onto the screen, the size of the grid, and the volume density of each spleen section. The differences in volume density of NE fibers among the three groups were analyzed by using a oneway analysis of variance (ANOVA). Factors reaching significance level of at least p <0.05 by A N O V A were subjected to

161 Newman-Keuls post hoc analysis to determine which groups contributed to the significant ANOVA.

High Performance Chromatography With Electrochemical Detection Middle pieces of spleen of approximately the same size in each animal were removed from liquid nitrogen, weighed rapidly, and placed in 0. I M sodium acetate buffer (pH 5). The internal standard, 3,4-dihydroxybenzylamine (DHBA), was added to give a final concentration of 0.25/xM and the sample was homogenized. The homogenates were centrifuged at 13,000xg for 10 minutes and the pellets were saved for protein assays (BioRad protein kit). The resultant supernatants were prepared for high performance liquid chromatography with electrochemical detection by adding 5 /~l ascorbic acid oxidase (25 U/ml) to 100 p.1 of supernatant to reduce the solvent front for optimal resolution of the NE peak. All samples for this study were prepared and run as a single group.

Data Analysis of LCEC Samples were analyzed for NE using a Waters model 510 solvent delivery system operated at a flow rate of 1.0 ml/min, a Waters 710B WISP automatic sample injector, a Biophase 5 /zm C18 reverse phase column and a glassy carbon thin layer electrochemical detector (TL-5, Bioanalytical Systems). The detector potential was set at 0.85 V (vs. an AgAgC1 reference electrode) using an LC-4B amperometric controller (Bioanalytical Systems). The buffer was 0.1 M citrate-0.1 M phosphate (pH 4.5) with sodium octyl sulfate (1.0-1.2 mM) used as the ion-pairing agent, and 1%22% methanol. The signal from the detector was recorded, peak heights and areas determined, and data analysis done using a Waters model 840 data and chromatography control station. Recovery was determined using D H B A as an internal standard. Samples corrected for recovery were compared with external standards to determine NE concentration, expressed as pMol NE/mg protein or g wet weight. The differences in NE levels among the three groups were analyzed using a one-way analysis of variance. Factors reaching significance by analysis of variance were subjected to Newman-Keuls post hoc analysis to determine which groups contributed to the significant ANOVA.

FACING PAGE FIG. 1. Dense plexus of varicose noradrenergic fibers (arrowheads) traveling along a trabeculum (T) in the spleen from an 8 month old F344 rat. Glyoxylic acid fluorescence histochemistry, x200. FIG. 2. Abundant noradrenergic varicosities surrounding the central artery (C) in the white pulp of the spleen from an 8 month old F344 rat. Many fluorescent linear profiles (large arrowheads) radiate away from this vascular plexus into the surrounding parenchyma of the periarteriolar lymphatic sheath. Some of these profiles lie in close apposition to yellow autofluorescent cells (small arrowheads). Glyoxylic acid fluorescence histochemistry. ×200. F1G. 3. Noradrenergic fibers (arrowheads) traveling along a trabeculum (T) in the spleen from a 27 month old non-neophobic F344 rat. Glyoxylic acid fluorescence histochemistry. ×200. FIG. 4. Noradrenergic varicosities (arrowheads) around the central artery (C) in the white pulp of the spleen from a 27 month old nonneophobic F344 rat. Glyoxylic acid fluorescence histochemistry, x200. FIG. 5. Noradrenergic fibers (arrowheads) coursing along a trabeculum (T) in the spleen from a 27 month old neophobic F344 rat. Glyoxylic acid fluorescence histochemistry, x200. FIG. 6. Vascular noradrenergic plexus (arrowheads) around the central artery in the white pulp of the spleen from a 27 month old neophobic F344 rat. Glyoxylic acid fluorescence histochemistry, x200.

F E L T E N ET AL.

162

Figure 7. Volume Density of NE Profiles per Spleen Section

RESULTS

Norepinephrine Fluorescence Histochemist~. Spleens of 8 month old rats displayed abundant noradrenergic innervation. Large plexuses of noradrenergic fibers coursed in association with the splenic artery, entered the spleen along the arterial branches, and continued either with large arteries as vascular plexuses, or within the splenic capsule and associated trabeculae, forming trabecular plexuses. Trabecular plexuses (Fig. 1) were composed of a high density of fluorescent varicosities that coursed along the edge of the trabeculae, occasionally in association with blood vessels within the trabeculae. These fibers continued into the white pulp. Small linear varicose profiles diverged from the trabecular plexuses into the surrounding parenchyma, primarily within the white pulp. The greatest density of noradrenergic fibers within the spleen traveled with vascular plexuses in association within the central artery of the white pulp and its branches (Fig. 2). Numerous small varicose linear and punctate profiles radiated from the vascular plexus into the surrounding periarteriolar parenchyma of the white pulp where they ended among lymphocytes (primarily T lymphocytes) of the periarteriolar lymphatic sheath (PALS). Linear chains of varicosities also coursed alongside clusters of autofluorescent cells within the white pulp (Fig. 2). In the white pulp, noradrenergic fibers were confined largely to non-nodular regions and extended to the inner edge of the marginal zone as well as the parafollicular zone; only very sparse distribution of noradrenergic varicosities was noted in the red pulp. In aged " n o r m a l " rats, noradrenergic innervation of the spleen was markedly decreased compared with the 8 month old adults. The trabecular plexuses (Fig. 3) demonstrated a much lower density of noradrenergic fibers, and the overall intensity of fluorescence in the remaining varicosities appeared reduced. Varicosities associated with blood vessels, especially the central artery of the white pulp and its branches (Fig. 4), were present but diminished in density and in overall intensity of fluorescence; fewer linear and punctate parenchymal fibers were present in the white pulp (Fig. 4). The decrease in varicosity intensity was evident in all aged spleens, rather than appearing randomly as would be expected if variability in the histofluorescence method was the cause. A greater number of yellow autofluorescent cells in the spleens of both groups of aged rats was observed. Parenchymal noradrenergic fibers often formed a close association with clusters of yellow fluorescent cells in these aging spleens. The white pulp was diminished in size and was often difficult to separate clearly from the red pulp. Noradrenergic innervation of spleen from aged neophobic rats appeared qualitatively similar to the aged " n o r m a l " animals described above (Figs. 5, 6).

0,06

0.05

0.04

0.03

002

!

0.01

r

0.(30 C

AG-NNP

AG-NP

FIG. 7. Volume density of noradrenergic terminals per spleen section in control (C; 0.0493-+0.0037), non-neophobic aged {AG-NNP; 0.0108+_0.005t and neophobic aged {AG-NP; 0.0096+_0.0021) rat spleen sections. Results are expressed as volume density % (o~ of section occupied by NE terminals). Significant difference from control Ip<0.01) is indicated by * TABLE 1 MEAN SPLENIC NOREPINEPHRINE LEVELS

Group

N

pMol NE/g Tissue (+SEM)

pMol NE/mg Protein (.+_SEM)

Control Aged Normal Aged Neophobic

6 6

76.00 + 4.86 38.47 + 9.21

1.96 _+ 0.14 0.98 +_ 0.25

6

31.56 + 4.76

0.92 + 0.17

Analysis of LCEC Data One-way analysis of variance revealed a significant group effect [F(2,15)= 13.02, p<0.01, pMol NE/mg wet weight tissue; F(2,15)=9.19, p<0.01, pMol NE/mg protein] (Table 1). Newman-Keuls post hoc comparisons revealed that splenic NE content was significantly diminished in both the aged non-neophobic rats (p<0.01) and the aged neophobic rats (p<0.01) compared with young adult rats. There was no significant difference in splenic NE levels in the aged neophobic compared with aged " n o r m a l " animals (,o<0.05). One-way analysis of variance demonstrated no significant difference in protein levels among the groups, F(2,15)= 1.19, p>0.05 (mg protein/tissue weight).

Morphometric Analysis Quantitation of NE terminals using point counting methods to give volume densities verified the descriptive impression of decreases in NE terminals. The volume density % of NE terminals per cross section of spleen was significantly decreased, F(2,6)=82.998,p<0.01 (volume density % per spleen section) in both normal aged and gustatory neophobic aged rats when compared with controls (Fig. 7). There was no significant difference between aged nonneophobic and aged neophobic rats.

DISCUSSION

The noradrenergic innervation pattern of spleens from young adult rats was consistent with previous studies of splenic noradrenergic innervation [16, 17, 38, 49, 501, and followed similar patterns of distribution of noradrenergic fibers in lymph nodes [15,16], gut-associated lymphoid tissue [14,25], and other lymphoid tissue [45]. With age, several changes in the distribution pattern of noradrenergic fibers in the spleen were observed. The morphology of the spleen

S P L E N I C N E I N N E R V A T I O N : A N A G I N G STUDY appeared different; the most notable difference was the indistinct boundary between red and white pulp and the reduced size of the white pulp. These findings are consistent with the age-related changes in goat spleens observed by Saigal et al. [40]. While the relative compartmentalization of noradrenergic fibers in specific regions of the spleen was similar in spleens from aged animals compared with their younger counterparts, noradrenergic fluorescent profiles were less abundant in the aged spleens, in agreement with the preliminary report of Capocelli et al. [11] in aged C57BL/6 mice. In addition, the overall intensity of fluorescence was diminished, suggesting a diminution in norepinephrine levels within these varicosities as demonstrated in the present study with LCEC. There was no significant difference in the levels of N E between the two aged subpopulations, divided on the basis of avoidance behavior to a novel gustatory stimulus. Splenic noradrenergic levels were decreased by 49.38_+12.12 and 58.47-+6.26% in the aged " n o r m a l " and aged " n e o p h o b i c " animals, respectively (based upon mean NE/g tissue wet weight -+SE). Thus, even though the neophobic behavior may be associated with diminished central levels of NE, it appears not to be associated with diminished peripheral levels of NE in the spleen. This finding suggests that central and peripheral NE systems may undergo age-related changes along different time courses, and may result from different mechanisms. NE released from nerve terminals can diffuse a considerable distance from its release site to elicit an effect upon target tissues [43]. A variety of cell types of the immune system, i.e., T ~nd B lymphocytes, macrophages, granulocytes and bone marrow stem cells, possess receptors for monoamine neurotransmitters ([5, 7, 20, 26, 30, 35, 41, 52]; recently reviewed by [21,22]). In vivo and in vitro administration of alpha- and beta-adrenergic compounds can affect the number of functions of some of these cell populations ([6, 20, 42, 47]; see [29] for a review). The demonstration of sympathetic innervation of lymphoid tissue, a cellular target for noradrenergic action within lymphoid tissue, and the capacity for neurotransmitter signal reception by these immune cells provides a firm physiological basis for neural modulation of cells of the immune system. Recent evidence suggests that other biogenic amines, such as epinephrine and serotonin, as well as norepinephrine, may be naturally taken up by sympathetic nerve terminals and subsequently released upon appropriate neural stimulation to act upon target tissue as a classical neurotransmitter [3]. Thus, sympathetic nerve terminals that innervate both the vasculature and parenchyma of lymphoid tissue may provide monoamine neurotransmitters, in varying concentrations, or ratios, to cells of the immune system depending on the prior physiological state of the animal. Such a possibility emphasizes the importance of understanding both the spatial relationship between nerve terminals and distinct lymphoid cell types, and the temporal events in the microenvironment that influence the concentration of different biogenic amines available for uptake and subsequent release. Once released, catecholamines may influence blood flow through the lymphoid organ, mediate ligand-receptor interactions on lymphocytes or other cells of the immune system, or act upon immune cells to alter release of secretory products. Catecholamines also may act in concert with other neurotransmitters, peptides and hormones. Several studies have demonstrated alteration in immune function following sympathetic denervation of the spleen,

163 either by surgical means, or, more commonly, with 6-OHDA [4, 23, 28, 51], although the direction of the change is not consistent and the basis for changes in immune reactivity is not clear. In general, it appears that enhanced antibody responses occur following neonatal sympathectomy with 6-OHDA and surgical denervation of spleen in adults, while acute denervation with 6-OHDA in adults results in suppression or no change in plaque forming cell (PFC) response. Interpretation of these results is difficult due to inherent problems in both denervation techniques. Neonatally induced alterations in immune function may be the result of both profound central and peripheral noradrenergic denervation [27], or the result of altered development of the immune system. Likewise, surgical denervation of the nerve supply to the spleen also may ablate other neurotransmitter inputs to the spleen, including a variety of peptides; the resultant increase in antibody response may be due to a depletion of both NE and other neurotransmitter systems that innervate the spleen via the same anatomical nerve trunks. In addition, the stress of sham surgery, used as a control for surgical denervation, results in a significantly diminished baseline of immune response with which to compare responses of denervated experimental animals. The finding of reduced immune responses in adults treated acutely with 6-OHDA [29] suggests that sympathetic innervation may be necessary for full immunocompetence. In support of this hypothesis, Hirsch et al. [24] have demonstrated that both the production and activity of interleukin-1 and -2 in vitro are significantly modulated by autonomic stimulation in vivo. Studies of aging humans, guinea pigs, hamsters, rats and mice have revealed that certain normal immune functions, primarily those involving the T cell responses, decline with age [31, 32, 46], concomitant with the decline in the peripheral noradrenergic system. Age-related immunologic dysfunction is associated with an increased incidence of autoimmune and immune complex diseases, certain types of cancer, and viral and fungal infections. Alterations in T cell mediated functions associated with aging include: (1) decreased T cell-dependent humoral immune response; (2) delayed skin aUograft rejection time; (3) reduced intensity of delayed hypersensitivity; (4) lowered resistance to tumor cell challenge; (5) decreased graft-vs.-host reactivity; (6) decreased cytolytic immune response; and (7) decreased proliferative responsiveness of T cells to mitogens ([31, 32, 34, 46]; reviewed by [33,48]). Impairment of the immune capacity may be the result of changes in the immune cells, alterations in their microenvironment, or, more likely, a combination of both. Several studies [1, 36, 37] support the idea that both cellular and extracellular age-related changes affect the decline in immune response, but that the majority of the age-related changes can be attributed to alterations in immune cells (primarily T cells) per se. The demonstration of noradrenergic fibers that exit vascular plexuses to end preferentially among fields of T lymphocytes provides a means for neural modulation, possibly through an effect upon T cells. The fact that the normal aging process is associated with both a decline in immunocompetence and a decline in splenic norepinephrine makes it tempting to hypothesize a causal relationship. Conversely, noradrenergic denervation may be compensatory rather than causal to immune dysfunction. However, preliminary examination of noradrenergic innervation of the murine spleen following lymphocyte depletion over a short time course using hydrocortisone or cyclophosphamide does not support the latter hypothesis.

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SPLENIC NE INNERVATION:

AN AGING STUDY

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