Plasticity of innervation of the medulla of axillary lymph nodes in the rat after antigenic stimulation.код для вставкиСкачать
THE ANATOMICAL RECORD 238:213-224 (1994) Plasticity of Innervation of the Medulla of Axillary Lymph Nodes in the Rat After Antigenic Stimulation G.E.K. NOVOTNY, T. HEUER, A. SCHOTTELNDREIER, AND c. FLEISGARTEN Department of Neuroanatomy, Dusseldorf University, Dusseldorf, Germany ABSTRACT The purpose of this investigation was to test the hypothesis that activation of the immune system in rats will lead to changes in the density of innervation in lymph nodes. In order to reduce the variability between animals, the rats were reared under sterile conditions and immunostimulation was effected by subcutaneous application of bovine albumin in a region draining to the axillary lymph nodes of both sides. Control animals received an equivalent application of sterile physiological saline. The animals were sacrificed 10 days and 27 days and 4 months after immunostimulation. The nerves in the axillary lymph nodes were quantified by light microscopy in silver impregnated sections and at the ultrastructural level on ultrathin sections. The survival times were chosen so that the first group was in the ascending phase of antibody production, the second group at the peak, and the third group in the declining phase. Both at the light and ultrastructural levels, there were statistically significant differences in the density of innervation of medulla between the groups, with a particularly pronounced increase in the group 4 months after immunostimulation. At the ultrastructural level, there was also an increase in the density of incompletely ensheathed axonal profiles in the parenchyma of the medulla, while the nerves associated with blood vessels were not increased. We conclude that immunostimulation leads to morphological changes in the innervation of the medulla of axillary lymph nodes, that are consistent with the concept of functional activation of the autonomic nervous system through the immune system. o 1% Wiley-Liss, Inc. Key words: Lymph node, Innervation, Immunostimulation, Silver impregnation, Ultrastructure In recent years the question of interaction between the immune and nervous systems has aroused interest. The majority of workers in this field assume a humoral axis from the central nervous system to the immune competent organs, as first postulated by Besedovsky (Besedovsky et al., 1988). However, several groups have also investigated the possibility of a direct innervation of immune competent organs (see review by Felten and Felten, 1991). Alterations in the density of innervation of immune competent organs with age have been reported (Ackerman et al., 1991a; Bellinger et al., 1987, 1988; Novotny et al., 19931, and deficits in the immune response have been shown after sympathectomy with 6-hydroxy dopamine (6-OHDA) (Ackerman et al., 1991b). Rearrangement of the innervation within a given compartment with maintenance of the total nerve content was reported for the spleen after drug-induced immunosuppression (Carlson et al., 1987). All these observations present suggestive evidence for a functional link between the peripheral innervation of lymphatic organs and the immune response. However, none of these investigations are conclusive because: (1) little is known about age changes in the peripheral innervation in general; (2) 0 1994 WILEY-LISS, INC global sympathectomy with 6-OHDA may well affect the immune response by unspecific mechanisms or via the central nervous system; and (3) the alterations in the spleen may be related to volume changes within compartments after lymphocyte depletion. Thus, an effect on the immune response through humoral mechanisms via the hypothalamic-adrenal cortex axis, as postulated by Besedovsky et al. (19881, still remains the most likely mechanism for immune regulation. Functional plasticity is documented in the central nervous system of invertebrates (Alkon et al., 1990) and higher mammals (Keller et al., 19901, and alterations in the size of the fields of innervation have been described in the peripheral nervous system after partial denervation (Terenghi et al., 1986; Navarro and Kennedy, 1988; Taylor et al., 1988). If there is a functional link between the peripheral Received February 2, 1993; accepted September 10, 1993. Address reprint requests to G.E.K. Novotny, Ph.D., Abteilung Neuroanatomie, Zentrum fur Anatomie und Hirnforschung, Med. Einrichtungen der Universitat Diisseldorf, Postfach 10 10 07, D-40001 Diisseldorf, Germany. 214 G.E.K. NOVOTNY E T AL. Diagram 1: Experimental Design GROUPS DURATION IN DAYS 0 10 2027 120 10 day stim. (N=10) 27 day stim (N=10) 4 mo. stim. (N=10) Control (N=10) 4 mo. cont. (N=4) Diagram 1. Diagrammatic presentation of treatment of the five animal groups. All animals were 3 months old at the start of the experiment (day 0 ) . = subcutaneous injection, Ag = antigen, V = Vehicle without antigen. The termination of the line indicates the time of sacrifice. + innervation of immune competent organs and their immunological function, one could expect to find morphological changes of the innervation in response to extremes of immunological activity. One problem to be anticipated in such a study is that each individual animal of a group is subject to an individual immunological history. If antigenic stimulation affects the innervation, then the individual history may well lead to considerable differences in the innervation between “normal” animals, which may be expected to obscure small but consistent differences due to a single antigenic exposure with experimentally practicable group sizes. To eliminate such a possible source of disturbance, this study was performed on animals reared under absolutely sterile conditions. The present publication reports on the light microscopical part of this study and presents supporting evidence at the electron microscopical level from a study designed to investigate ultrastructural changes under such conditons. MATERIALS AND METHODS Animals Forty-four male HAN-Wistar rats were used. The animals were bred and reared under sterile conditions in isolation units in the Tierversuchsanlage of Dusseldorf University according to the regulations for housing of experimental animals. Sterilized food and water were available ad libitum. Because of the limited capacity of the isolation units, the animals were reared and experimentally treated in eleven groups of four siblings each. Ten of the sibling groups each contained one control animal and one animal of each of the three experimental groups. The eleventh group consisted of four long-term controls that remained untreated. At the start of the investigation all animals were 3 months old. prising 10 animals and three experimental groups also of 10 animals each. The first experimental group received one application of antigen (see below) and was sacrificed 10 days later (10 day stimulated). The second group received two applications of antigen a t an interval of 20 days and was sacrificed 27 days after the first application (27 day stimulated). The third group received identical treatment to the second group and was sacrificed 4 months after the first antigen application (4 month stimulated). The selected schedules and survival times ensure that the first group is in the ascending phase of antibody production, the second group is a t the peak of antibody production, while the third group is in the declining phase (Herbert, 1968). The control group was matched with the second group a t the maximum of antibody production and received injections of physiological saline at the same intervals. The fifth group, added after another study in our laboratory had revealed age effects on the innervation of lymph nodes in the rat, consisted of only 4 animals as controls for the long-term (4months) survival group. The animals remained untreated and were sacrificed a t the same age as the long-term survival group (4months control). lmmunostimulation Immunostimulation was achieved with the subcutaneous injection of purified bovine albumin (Serva) in incomplete Freund’s adjuvans (Sigma Chemical Co., St. Louis, MO). One hundred microliters of this mixture, containing 100 pg albumin and 50 p1 incomplete Freund’s adjuvant in sterile physiological saline, were applied interscapularly. This ensured effective stimulation of an antigenic response with the appearance of germinal centers and plasma cells in the axillary lymph nodes (Figs. 1-4). Control injections consisted of 100 ~1 sterile physiological saline. Observation of the animals after application of antigen did not give any Experimental Design indication of adverse effects, except in one animal The animals were divided into five groups which which developed a slight granulomatous reaction at were treated as summarized in Diagram 1. The pri- the site of injection. All animals survived the injections mary experiment consisted of one control group com- for the predetermined period till sacrifice for perfusion. LYMPH NODE IMMUNOSTIMULATION Figs. 1-4. Micrographs of semithin sections of axillary lymph nodes from control and experimental rats to illustrate the effect of immunostimulation. All calibrations 50 km. Fig. I . Lymph node from a control animal, showing the homoge- neous cortex (C) and the medulla (M) exhibiting many narrow vascular channels. x 70. Fig. 2. Lymph node from a n animal 27 days after immunostimulation. Two germinal centers (arrows) are visible in the cortex (C). The Light microscopy Histological Procedures All animals were sacrificed by transcardial perfusion with a mixture of 1.25% glutaraldehyde and 1%paraformaldehyde in 0.1 M phosphate buffer of pH 7.4 at 0°C under deep anaesthesia (total areflexia) after intraperitoneal application of a lethal dose of 36% urethane. The initial perfusion pressure of 120 mm Hg was reduced by 20 mm Hg a t 10-minute intervals, with a total perfusion time of 30 minutes. 215 medulla (M)contains abundant wide vascular channels, many of which are lymphatic. X 70. Fig. 3. Lymph node from a n animal 4 months after immunostimulation. Plasma cells (arrows) are present in the medulla. x 600. Fig. 4. Lymph node from a n animal 27 days after immunostimulation. Detail of a germinal center, showing mitotic cells (arrows). x 500. The axillary lymph nodes were removed immediately after the perfusion was completed and post-fixed in 10% formaldehyde solution for 4 weeks. To avoid damage to the lymph nodes, which were embedded as a group in the adipose tissue of the axilla, they were only separated from the fat sufficiently for identification and isolation from each other. Thus, each tissue block also contained some adipose tissue, and for this reason it was not deemed practicable to determine the volumes or weights of the nodes. Subsequently, the lymph 216 G.E.K. NOVOTNY ET AL. nodes were dehydrated over ethanol and embedded in paraplast (Monoject Scientific Inc., Athy, Co. Kildare, Ireland). One lymph node was chosen from each animal and serial sections were cut at 10 pm, mounted on slides, and silver impregnated as described (Novotny and Gommert-Novotny, 1988). Out of each of these series, three slides (6 sections each) were taken from each third of the series and stained with hematoxylin and eosin or cresyl violet for the precise determination of the presence of germinal centers or plasma cells, as a control on the actual immune stimulation, germinal centers being absent in non-stimulated animals, while plasma cells are exceedingly rare. Electron microscopy For ultrastructural investigation, the whole lymph nodes, excised from the animals, as described for light microscopy, were post-fixed in 1%osmium tetroxide for 3 hours, dehydrated over acetone, and embedded in Spurr’s medium. One lymph node was chosen from each animal and completely sectioned, whereby ten ultrathin serial sections were gathered on 100 mesh copper grids at intervals of 100 pm throughout the total extent of each lymph node. Semithin sections were collected for each group of ultrathin sections, to enable comparisons between light and electron microscopy, and to control for effective immunostimulation by the presence of germinal centers and plasma cells. The ultrathin sections, contrasted with uranyl acetate and lead citrate, were viewed in a Zeiss EM9 or Hitachi H-600 at 60 or 75 kV and a primary magnification of 15,000 or 20,000. Morphometric Procedure Light microscopy In a previous investigation on the density of innervation of lymph nodes (Kliche and Novotny, 1987) every 6th section was evaluated. By random selection of sections from this existing pool, we determined that it is sufficient to evaluate every 24th section only. To reduce the variability in the present study, the sections were not chosen totally randomly, but were selected randomly in equal numbers from each third of the lymph node. If the number of sections of the total lymph node was fewer than necessary t o arrive a t a total of 6 sections (e.g., 100 sections = 100/24 = 4.167), then 6 sections were selected (i.e., 2 from each third); otherwise the next full number of the computed figure was taken (i.e., 10 sections if the computed number was 9.3). Each section was completely scanned in a meander pattern under oil immersion optics ( x 800) and the length of all nerves encountered within the lymph node (excluding the capsule) was measured under direct microscopic vision using a Zeiss Morphomat 10 digitalization tableau system. Additionally, the composition of each microscope field (i.e., cortex or medulla) was noted and the location of the nerves within these compartments was recorded. The total number of fields gave the precise measure of the area of each section and thus, together with the known section thickness, enabled the computation of the sampled volume of each section. Summation of all the sections analyzed in one animal gave the total volume sampled. The occurrence of branching of the axons was also registered. Measures were taken to determine whether the in- vestigated lymph node was immunostimulated using additional stains. This was in fact superfluous, since the silver impregnated sections clearly revealed the immunological status of the lymph node, so that the observer could not fail to know to which group of animals a given lymph node belonged. For this reason it was not possible to perform a blind morphometric analysis. However, to reduce observer bias no computations whatever were performed on the raw data until after all measurements for all groups were completed. Electron microscopy One lymph node from each animal was selected for electron microscopy and completely sectioned. Series of 10 ultrathin sections were collected a t intervals of 100 pm. Between ultrathin series, semithin sections were collected and stained with toluidin blue. These semithin sections were intended as controls, to ascertain the immune reaction of the lymph node. In actual practice this was superfluous, since the immune reaction was also obvious on the ultrathin sections. Only 1 section from each of the 10 sections comprising an ultrathin series was chosen randomly and completely searched for axonal sections, grid square by grid square. Series for sampling were selected in a semi-random fashion to ensure that all portions of the lymph node were equally represented. Sampling from series was continued until a total count of at least 150 incompletely ensheathed axonal sections (varicosities) was attained for each lymph node. The purpose of this procedure was to obtain quantitative data on the percentage representation of various categories of varicosities and their relation to cellular elements. These results will be presented in detail elsewhere. Together with the aforementioned data, information was gathered on all axonal sections, including their location in cortex or medulla, as well as the total area of lymph node searched. From this evaluation it is possible to derive information on the density of innervation and these results are presented in the present publication. Sampling of each individual lymph node was performed by four observers independently, with each observer aiming to contribute approximately equal proportions of the total count. Since termination of the count with the attainment of the required figure will cause considerable bias in the number of axonal sections per unit area, each observer completed the evaluation of the section containing the axonal section number aimed at. Thus, usually more than the requisite number of axonal sections was counted, and the summation over four observers lead to totals of more than 150 axonal sections in almost all instances. Again, the raw data was not analyzed until all the data had been gathered, in order to reduce possible observer bias. As the counting of a representative number of axons at the ultrastructural level is exceedingly laborious, only those groups were evaluated that had proved of interest a t the light microscopical level, i.e., both control groups and the 27 day and 4 month stimulated groups. This decision was taken by one author (G.E.K.N) and the remaining observers were not informed as to the result of the light microscopic investigation until the gathering of the ultrastructural data was completed. LYMPH NODE IMMUNOSTIMULATION Statistical Procedures The various parameters obtained were compared between the groups using the Kruskal-Wallace non-parametric analysis of variance (H-test) a t a significance level of P < 0.05. Global comparisons attaining this significance level were further analyzed using the comparison of pairs according to Schaich & Hamerle (Bortz et al., 1990). RESULTS General Obsewations Light microscopy Lymph nodes from non-immunostimulated control animals show a homogeneous cortex and a medulla lacking prominent vascular channels or plasma cells (Fig. 1). All axillary lymph nodes from animals surviving the application of antigen by 10 or 27 days before sacrifice contained germinal centers (Figs. 2,4). Four months after immunostimulation germinal centers could only be found in few lymph nodes. However, plasma cells could be located in the medulla in numbers not present in the control animals (Fig. 3). Nerves were present in the medulla of all lymph nodes examined. However, 4 months after immunostimulation their number seems to be distinctly increased and branching of the nerves is more frequent (Figs. 5,6). Electron microscopy As already noted at the LM level, immunostimulated lymph nodes contained germinal centers and large numbers of plasma cells, which were also obvious a t the ultrastructural level. Nerve bundles could be found throughout the medulla and on the borderline between medulla and cortex. The remaining cortex and all germinal centers were devoid of innervation. The axons within the nerves were either completely ensheathed by Schwann cell processes (axonal sections), or partially denuded (varicosities) (Figs. 7-9). Some axons were myelinated. Both varicosities or axonal sections could contain vesicles, but vesicles were never found in myelinated axons. As previously described for normal animals (Novotny, S988), varicosities were predominantly associated with reticular cells (Figs. 7,9). Of the lymphocytic population, plasma cells were most frequently related to varicosities (Fig. 81, and associations of varicosities with lymphocytes (which were not further characterized) were rare. Quantitative Results Volume changes Since a fixed proportion of the sections of each lymph node was analyzed at the light microscopical level, the actually scanned volume of each lymph node is approximately proportional to its total volume. The result is presented graphically in Figure 10 and demonstrates an increase in the analyzed total volume of stimulated lymph nodes 10 days after the first antigen application, which is statistically not significant (P = 0.2700). There is a subsequent statistically not significant decline to below control values in the 4 month survival group. As may be further noted, the alteration in analyzed volumes is solely due to changes in the volume of 217 the cortex, the medullary volume remaining remarkably constant with only an insignificant increase in the 4 month immunostimulated group (P = 0.6560). The volume changes in the cortex do not attain statistical significance (P = 0.0953) mainly because of scatter, indicated by the large standard deviations, reflecting considerable differences in the size of the randomly selected lymph nodes from individual animals. The relation between volume of cortex and volume of the medulla is demonstrated in Figure 11. The ratio of cortex to medulla increases up to 27 days after antigenic stimulation and declines after longer intervals. Here there is a significant difference in global comparison (P = 0.0018) and there are significant pair differences between the 4 month stimulated group on the one hand and the 10 day and 27 day immunostimulated groups on the other (both with P < 0.05). This is due to the relatively constant relationship between medulla and cortex within each group, despite large differences in total size of the lymph nodes. The time course of the change in the ratio of the volumes roughly corresponds to that of the production of antibodies by our immunization schedule, as based upon data in the literature (Herbert, 1968; Harlow and Lane, 1988). Innervation As shown in Figure 12, the average total length of nerves measured per lymph node in each group increases from the control to the 4 month survival group, but is lowest in the 4 month control group. The total length of nerves in the whole lymph node, as well as the total values for cortex and medulla, all show significant differences in global comparison (total: P = 0.0053; medulla: P = 0.0022; cortex: P = 0.0114). In post-test pair comparison, there is no significant pair difference for the cortex, for the total lymph node there is a significant pair difference between the 10 day and 4 month stimulated groups (P < 0.011, and in the medulla there is a significant pair difference between the control and 4 month stimulated groups (P< 0.05) and between the 10 day stimulated and 4 month stimulated groups (P < 0.0s). When related to the total volume as density of innervation, there is an increase in the groups 27 days and 4 months after antigenic stimulation, with a slight decrease in the 10 day group (Fig. 13). The groups differ significantly in global comparison (P = 0.00009), with significant pair differences (P < 0.05) between the control, 10 day stimulated, and 27 day stimulated groups on the one hand and the 4 month stimulated group on the other. There is no significant difference between the 4 month stimulated group and the 4 month control group or between the latter and the original control group, which may be attributed to the few animals available for the 4 month control group. For the innervation of the medulla the global difference is equally pronounced (P = 0.00008), with significant pair differences between the control, SO day stimulated, and 4 month control groups vs. the 4 month stimulated group. There is no significant difference for the medullary innervation between the short-term control group and the 4 month control group. There is practically no innervation of the cortex, the few nerves located in this compartment are close to the border of the medulla and are of no consequence to the 218 G.E.K. NOVOTNY ET AL. Figs. 5-6 LYMPH NODE IMMUNOSTIMULATION Figs. 7-9. Electron micrographs illustrating axons and varicosities with their typical relationships to cells. All calibrations 1 pm. Fig. 7. A nerve within a group of plasma cells in a lymph node of an animal 27 days after immunostimulation. There are two varicosities (arrows),the upper of which is associated with a plasma cell, while the lower one (possibly sensory) is approximated to the process of a reticular cell. The third profile (arrowhead) is completely ensheathed and thus axonal. x 16,000. Figs. 5-6. Silver impregnated sections from control and experimen. tal animals to show nerves. All calibrations 50 pm. Fig. 5. Lymph node from control animal. A representative field from the medulla showing two nerves in focus (arrows). The arrowheads indicate the position of nerves present in the visual field, but out of the focal plane at this setting. Note that none of the nerves branch. x 500. Fig. 6. Lymph node from an animal 4 months after immunostimulation. An equivalent field of the medulla to that shown in Figure 5, with nerves (small arows) and branching of nerves (large arrows). Note the distinct increase in density of innervation after 4 months of immunostimulation. x 500. 219 Fig. 8. Animal 27 days after immunostimulation. A fine nerve accompanying a blood vessel (bottom of illustration), with two varicosities containing vesicles (arrows). The upper varicosity is closely apposed to a plasma cell, whereas the lower one is not closely associated with any cell. x 20,000. Fig. 9. Animal 4 months after immunostimulation. A nerve containing two axons (arrowheads) and three varicosities farrows),the one on the left with vesicles. All of the varicosities are associated with reticular cells. x 12,000. cortex as a whole. The innervation of the cortex shows a marginally significant difference in the global comparison between the groups (P = 0.050991,with no significant pair differences. It should be noted that the density of innervation increases for the total lymph node and the medulla, while it decreases for the cortex. As shown in Figure 14, the frequency of axonal branching per unit volume of medulla is distinctly increased in the 4 month stimulated group. The global group comparison is very significant (P = 0.0024), the 220 G.E.K. NOVOTNY ET AL. Average Volumes per Lymph Node in all Groups (Actual volumes analyzed) ri Average Length of Nerves Measured 10000 [zlTotal Cortex Medulla L m' E 8000 l .r [71 control @$ 1Od. stirn. [IIII 27d. stirn. 6000 A .wZ rn f 4000 2000 0 Groups Control 10d. stim. 27d. stim. 4mO. stim. 4mO. cont. Groups Figs. 10-20. Bar diagrams showing quantitative changes in lymph nodes after immunostimulation. The error bars show the standard deviations. Significance of pair comparisons in post-test, after global comparison attaining P < 0.05, is indicated by identical symbols above related bars. One symbol indicates P < 0.05, two symbols indicate P < 0.01, and three symbols indicate P < 0.001. Fig. 10. Diagrammatic representation of the average volume of lymph node analyzed at the light microscopic level in each group of animals, with a fixed proportion of sections selected for each lymph node. Thus, the volumes shown give a n approximate estimate of the relative volumes of the actual lymph nodes. In addition to the total volumes, the volumes for cortex and medulla are also indicated. Note the minimal change in the volume of medulla analyzed in all the groups. There are no significant differences. Fig. 12. Showing the average total length of nerves measured per lymph node at the light microscopic level in all animal groups. Nerve Length/Vohe for all Groups 400F 3001 Ratio CwCx/Medulla :i- i i 8 0 .r m Iy 0 5 0Control 10d. stim. Control 10d. stim. 27d. stim. 4mo. stim. 4mo. cont Groups m27d. stim. memo. stim. m4rno. cont. 4 3 2 Fig. 13. Light microscopic density of nerves in the various compartments of axillary lymph nodes in all animal groups. Abbreviations: NN, = nerve density per total lymph node volume; NN, = nerve density related to volume of cortex; N/V, = density of innervation in medulla. There is a distinct increase in the innervation for the total lymph node and the medulla 4 months after immunostimulation, whereas the density of innervation in the cortex is unchanged over all groups. 1 0 Groups Fig. 11. Diagram showing the changes in the average ratio between cortex and medulla for each group at the light microscopic level. Note the increase up to the 27 day immunostimulated group and the distinct decline to the 4 month immunostimulated group. The time course of these volume changes parallels the time course of changes in antibody production. However, it should be noted that the antibody production 4 months after immunostimulation is still considerably elevated over control levels. pair difference between the 4 month stimulated and 4 month control group attains P < 0.01 and the difference between the 4 month stimulated and 10 days stimulated groups is also significant (P < 0.05). Even when related to the length of nerves in the medulla, the increase in branching remains significant (Fig. 15) with P = 0.032, but no single pair difference attains significance. A slightly higher significance is obtained for axonal branching in the total lymph node (branches per unit length nerve: P = 0.0252), since the values for the cortex serve to amplify the effects (not shown). Electron microscopy The areas of the lymph nodes analyzed at the electron microscopical level are shown in Figure 16 for all groups. Since the aim was to register a certain minimum number of varicosities per animal, the area of lymph node analyzed roughly reflects the density of varicosities. The relationship is not quite precise, since the independent activity of four observers, each completing the evaluation of the section containing the varicosity number to be attained for the individual quota, resulted in variably higher total counts. The analyzed total areas differ significantly in global com- 221 LYMPH NODE IMMUNOSTIMULATION Nerve Branches per Unit Volume Medulla EM Areas Analyzed in Groups /Total Control “E E T l.5i L I “ I ln 1.0 T area Medulla N E u 4 m o stim € 3 .-C m E 2 a 1 0 Groups Control Fig. 14. Showing the frequency of nerve branching per unit volume medulla at the light microscopic level for all groups. There is a marked increase in branching 4 months after immunostimulation. 5r Nerve Branching in all Groups -r 1 CJ control 1Od. stim. 27d. stim. 4mo. stim. 4mo. cont. Groups Fig. 16. Graphic presentation of the average total area of ultrathin sections analyzed for each group, together with the distribution of the area into cortex and medulla. Nerves in Medulla 27d. stim. L 3 01 a 111 2 0 2 C e m i 0 Groups Fig. 15. Nerve branching related to unit length of nerve in medulla at the light microscopic level for all groups. Even when related to unit length of nerve the global comparison of the groups attains the significance level, but none of the pair comparisons is significant. parison (P = 0.03181, with no significant pair differences. The areas of medulla analyzed differ more distinctly (P = 0.00047) with a significant pair difference only between the control group and the 4 month immunostimulated group, whereas the cortical areas do not differ significantly. At the electron microscopical level, nerves are found almost exclusively in the medulla. The few instances of axonal profiles located in the cortex are always close to the border of the medulla, in grid squares containing both cortex and medulla. In the following, the innervation is thus only related to the area of medulla analyzed. The number of nerve sections ( = bundles of axons) per unit area medulla is shown in Figure 17. Global comparison reveals a highly significant difference (P = 0.00172), with a significant increase between the control and 4 month immunostimulated groups. Significance is not attained between the 4 month control group and the 4 month immunostimulated group, which may be attributed to the small size of the 4 month control group. The number of axonal sections per unit area is shown in Figure 18. The results closely Groups Fig. 17. Showing the average density of nerves in the medulla of the lymph nodes of all animal groups at the electron microscopic level. The relations between the groups should be compared with those presented in Figure 13, derived at the light microscopic level, which are almost identical. Note that a “nerve” contains a variable number of axons, and that the number of nerves to be found a t the light microscopic level is considerably fewer than those identifiable at the electron microscopic level. resemble those for nerve sections, the global difference between groups giving a value of P = 0.0134. Again only the 4 month immunostimulated and control groups differ significantly. This is reflected in the almost constant number of axons per nerve over all groups (maximum = 4.5; minimum = 3.21). Nerves closely applied to blood vessels, i.e., with no intervening cell processes, were classified as vessel nerves. Figure 19 shows the incidence of these nerves in the medulla for all groups. Global comparison reveals a significant difference between groups (P = 0.016561, the difference being caused by the drastic reduction of such nerves in the 4 month control group with significant differences between this group and the 4 month and 27 day stimulated groups (P < 0.05). 222 G.E.K. NOVOTNY ET AI,. Control d m27d. stim. a m4mo. stim. +++ T > 400 0 $ 300 9) 0 a 2 7 d . stim. CzJ4mo. stim. 9) .-0L vl C E 0 X 200 100 0 Groups Groups Fig. 18. Graphic demonstration of the changes in the density of axons at the electron microscopic level in the medulla of the various groups. The increase in the number of axons after immunostimulation is not quite as pronounced as that of nerves, indicating that the average number of axons per nerve is slightly reduced. For the longterm control group, there is a slight increase in the number of axons per nerve, when compared with the younger control group. Fig. 20. Diagram showing the incidence of only partially ensheathed axonal sections (varicosities) for all groups of animals. The group of animals 4 months after immunostimulation exhibits a particularly pronounced increase of such axonal sections, when compared with the short-term control group. DISCUSSION d Nerves of Medulh V Control T m27d. stim. + a 4 m o . stim. Groups Fig. 19. Showing the average density of nerves accompanying blood vessels, and not separated from these by intervening cells processes, in the medulla of the lymph nodes of the various animal groups. Note that there is not such a n increase after immunostimulation, as was found for nerves not associated with blood vessels. However, the longterm control animals possess very few vessel nerves, so that the maintenance of this category of nerve in the 4 month immunostimulated group may reflect some activation of this class of nerves. The functionally relevant aspect of lymph node innervation may be presumed to be reflected by the nonensheathed axonal profiles, which we have defined as “varicosities,” irrespective of their content of synaptic vesicles. Figure 20 illustrates the incidence of varicosities in the medulla for all groups. Global comparison shows significant differences tP = 0.00064), with a highly significant difference between the 4 month immunostimulated and control groups (P < 0.001). The remaining differences do not attain the significance lev 4. The main finding in this study is an increase in the density of innervation in the medulla of axillary lymph nodes 4 months after immunostimulation, at both the light and the electron microscopical level. However, the total volumes analyzed at the light microscopical level, and hence also the volumes of the lymph nodes, are not identical; they are less in the 4 month group, although this difference is not statistically significant. The question therefore arises, whether the increased density of innervation is genuine, or a spurious effect, due to the reduction in volume, with the innervation simply maintained at its absolute value. Figure 10 shows the volumes of cortex and medulla scanned in all groups. It may be seen that the actually scanned volumes of the medulla, where the changes in density of innervation are most pronounced, are almost identical between the groups, with a slight tendency to the greatest volume in the 4 month immunostimulated group. At the ultrastructural level the areas analyzed differ significantly. However, this difference is simply a reflection of the increase in the number of varicosities, since this was the parameter determining the termination of the registration. It is thus evident that alterations in volume cannot be responsible for the increase in the density of innervation that is found in the medulla. The density of innervation of axillary lymph nodes in the rat is increased in animals aged over 2 years (Novotny et al., 1993). Our original control group was matched to the 27 day survival group, because the greatest effect on the innervation was anticipated at the height of antibody production. The 4 month survival animals are thus 3 months older than the controls. For this reason, a further control group, comprising only four animals, was introduced to match the long-term survival group in age. It is quite evident that this control group shows not the slightest tendency to an increase in the density of innervation. This finding LYMPH NODE IMMUNOSTIMULATION agrees well with the results of an unpublished pilot study (Krucken), where the young adult group of animals, aged approximately 4 months, also exhibited a slightly lower density of innervation than a juvenile group, although the aged animals (> 2 years) had the highest density of innervation. Together with the present results, this suggests that the density of innervation does not change appreciably during adolescence and young adulthood and only increases distinctly in old age. Interestingly, the values for innervation density, obtained by identical methods, are higher in the animals from a sterile environment than from normally reared animals (Novotny et al., 1993). However, comparison is difficult, because the ages are not identical, and we are currently engaged in determining the precise time course of age changes. A further problem of this study is the inability to conceal from the observers, from which group of animals each lymph node derived. At the light microscopic level, it is impossible to reliably identify nerves without actually focusing through a suspected structure in the microscope. Identification of all nerves on photographs is impossible. It would have been conceivable to let an observer evaluate a microscopic field chosen by another person, so that the whole section cannot be viewed, but this would have caused a drastic prolongment of an already dreary procedure. Also, the question of selection of a field of view would have arisen. Such a course is not practicable. At the electron microscopical level it would have been theoretically possible to take random photographs to be evaluated by another observer. However, the density of innervation is so low that many thousands of photographs would have to have been made, the vast majority of them without nerves. The time consumption and costs would have been prohibitive. In our view, there was no alternative to the procedure we have adopted, even if this entails the danger of introducing bias into the evaluation (for precautions see the Materials and Methods section). Additionally, in the ultrastructural investigation four observers obtained their results independently, with no knowledge of possible trends. Finally, the actual results obtained do not conform to those anticipated. As evidenced by the design of this experiment, the greatest effect was expected to be found a t the peak of antibody production, i.e., in the 27 day immunostimulated group. Maybe naively, anticipated changes were expected to revert to control values in the 4 month immunostimulated group. These expectations are clearly negated by the results. In addition, there is very good agreement between the light microscopic and the electron microscopic analysis (compare Figs. 13 and 17).The ratio of innervation density for the medulla between the controls and the 4 month stimulated group is 1:l.g for the light microscopic analysis and 1:2.1 at the electron microscopic level. It is hard to conceive the independent bias of four observers, without knowledge of the trends of the evaluation, producing such consistent results. We therefore conclude that the immunostimulation has produced a genuine increase in the density of innervation of the medulla of axillary lymph nodes 4 months after immunostimulation. The electron microscopical observations demonstrate that the increase in the density of innervation is accompanied by an 223 equally pronounced increase in the number of varicosities, which argues that the observed changes are functionally relevant. The time-course of the changes found is an important factor. Our immunization schedule was designed to produce a response maximum a t 27 days, with a gradual decline in antibody production to the 4 month immunostimulated group. If the innervation of the axillary lymph nodes serves to stimulate antibody production (which was our original assumption), then one would anticipate a pronounced increase in the innervation before the peak of antibody production is attained (i.e., during the phase of greatest acceleration of antibody production), or at least in synchrony with antibody production. However, the greatest change was actually observed after the peak of antibody production was passed. This means that the morphologically visible changes in the innervation lag behind the immunological events. We thus consider it to be unlikely that activation of the direct innervation of axillary lymph nodes is concerned with the mounting of an antigenic response in these nodes, unless the morphological changes take a long time to become visible. The first reports of neuronal plasticity suggest such a long time course (3-6 months) before morphological alterations can be registered (Raisman, 1969), but more recent studies have demonstrated recognizable changes after intervals of only 4-8 days (Taylor et al. 1988; Alkon et al. 1990). We are therefore inclined to believe that the lack of a pronounced effect 27 days after immunostimulation reflects the late response of the peripheral nervous system. Nevertheless, groups between 27 days and 4 months, as well as periods longer than 4 months, would have been of great interest. If the innervation of lymph nodes is not concerned with the stimulation of antigen production, the question arises as to what function may be attributed to it. The data in the literature are seemingly in conflict, which may be illustrated with just two examples. Stimulation of the sympathetic outflow leads to increased outflow of lymphocytes from popliteal lymph nodes (McHale and Thornbury, 1990), which may be classified as activation of the immune system. On the other hand, sympathectomy has been reported to enhance immune responses (Besedovsky et al., 19881, which may be interpreted as indicating an inhibitory action of the nervous system on the immune system. However, such conflicting reports may be reconciled. The first instance is an example of cellular mobilization, whereas the second example is of an inhibitory effect on antibody producing cells, i.e., the humoral immune response mediated by plasma cells. Seen in this light, the increase in innervation, which we have found within the plasma cell regions of the axillary lymph nodes, may reflect the activation of a negative feedback circuit concerned with the down-regulation of the humoral immune response. This hypothesis agrees well with two of our previous observations. Firstly, we have consistently observed nerves to be concentrated in plasma cell areas of lymph nodes, and ultrastructural axonal varicosities to be associated with reticular cells and plasma cells (Novotny and Niche, 1986; Novotny, 1988; Novotny et al., 1993). Secondly, there is a marked increase in the density of innervation of axillary lymph nodes in aged rats (Novotny et al., 19931, 224 G.E.K. NOVOTNY ET AL. and the reduced reactivity of the immune system in old age is well known. It thus seems most likely that for the lymphocytic cell population the innervation of axillary lymph nodes in the rat is primarily concerned with plasma cells and not with T lymphocytes. In this connection it is of interest that B cells have been shown to possess a higher density of nerve growth factor receptors (NGF-R) than T cells, and to be stimulated to IgM production via these receptors (Otten et al., 1989). Inflammatory stimuli significantly increase NGF production, and denervation has been shown to be accompanied by an increase in NGF levels, which are decreased by renewed innervation (Weskamp and Otten, 1987). It could thus be conceived that the innervation of lymph nodes is concerned in such a NGF mediated regulation of antibody production. However, the time course of events reported by Weskamp and Otten is in the range of hours, as compared to weeks for the events described here. A very high proportion of axonal varicosities is associated with fibroblastic reticular cells of the medulla (Novotny, 1988). In the medulla of lymph nodes these cells also possess NGF receptors (Thompson et al., 1989). It is thus possible that the innervation may be concerned with regulative mechanisms via these cells. 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