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Anterior hypothalamic stimulation decreases serum immunoglobulin G concentrations.

Neuroimmunological research has indicated that hypothalamic structures play an important role in the modulation of immunoresponsivity (Ader, Felten, & Cohen, 1991; Dunn, 1989; Madden & Felten, 1995). In addition, research in the area of Health Psychology has implicated hypothalamic structures as part of a system that may be important in modulating the reactions of the immune system to stress (Madden & Felten, 1995). The effects of stress, coping styles, psychosocial factors, and personality characteristics have been shown to affect an individual's ability to resist disease (O'Leary, 1990; Siegal, Rodin, Seligman, & Dwyer, 1991; Zakowski, Hall, & Baum, 1992).

For example, destruction of hypothalamic areas offered protection against lethal anaphylaxis and increases in tumor growth (Luparello, Stein, & Park, 1964; Kavetsky, Turkevich, Akimova, Khayetsky, & Matuechuck, 1969). Lesions of the anterior hypothalamus protected rats from the effects of experimental autoimmune encephalomyelitis (Abramsky, Wertman, Reches, Brenner, & Ovadia, 1987) and anterior lesions reduced the production of antibody to ovalbumin in rats (Tyrey & Nalbandov, 1972).

Neural areas outside of the hypothalamus, particularly those in the limbic system, have also been implicated in immune functions. For example, lesions of septal and hippocampal structures differentially effected IgG, IgA, and IgM production (Nance, Rayson, & Carr, 1987). Lesions of the fornix have been shown to increase the proliferation of splenic cells and hippocampal lesions have resulted in the increase of both splenic and thymocyte cells (Rozman, Cross, Brooks, & Markesbery, 1985).

Electrophysiological recordings have provided evidence for involvement of both anterior and medial aspects of the hypothalamus in immunoresponsiveness. Besedovsky and Sorkin (1977) demonstrated that medial hypothalamic neuronal firing rates increased in the face of antigenic challenge. This increase in firing rates corresponded to the formation of IgM class antibodies in the spleen of rats challenged with sheep red blood cells. Saphier, Abramsky, Mor, and Ovadia (1987) recorded neuronal activity from the anterior, preoptic, and paraventricular areas of the hypothalamus. These authors found that firing rates of neurons in the anterior and preoptic areas of the hypothalamus increased in the face of antigenic challenge with sheep red blood cells whereas activity in the paraventricular nucleus showed an initial decrease followed by an increase in firing rates.

Studies investigating effects of electrical stimulation of the hypothalamus on immunity are lacking. Fessel and Forsyth (1963) demonstrated that electrical stimulation of the lateral hypothalamus in rats increased gamma globulin levels for up to 10 days following brain stimulation. Lambert, Harrell, and Achterberg (1981) found that electrical stimulation of the ventromedial nucleus reduced the capacity of the reticuloendothelial system to remove antigen from the blood stream of rats.

The effect of hypothalamic stimulation on immune functions and particularly on changes in specific classes of antibodies needs to be continued. The research of Fessel and Forsyth (1966) analyzed total gamma globulin but did not indicate what class(es) of antibodies were affected (e.g., IgG, IgD, IgM, IgE, IgA). In order to investigate hypothalamic influence on a specific class of antibody the present study measured the effect of electrical brain stimulation on the concentration of Immunoglobulin G (IgG) in rat serum.

Materials and Methods

Subjects

Subjects were 24 experimentally naive male, albino Sprague-Dawley rats (ARS Sprague-Dawley, Madison, Wisconsin) weighing 250-300 grams at the beginning of the experiment. All animals were individually housed and allowed food and water ad libitum throughout the experiment.

Apparatus

During intracranial stimulation, subjects were placed in a Gerbrands operant chamber with a hole in the ceiling to permit passage of a shielded cable. Dimensions of the chamber were 38.58 cm x 21.59 cm x 21.59 cm. The floor consisted of stainless steel bars, and the walls and ceilings were constructed of Plexiglas (Ralph Gerbrands, Arlington, Maryland). Brain stimulation was delivered by two Grass S48 brain stimulators (Grass Instruments, Quincy, Massachusetts). A bifurcated shielded cable 120 cm in length with a stainless steel spring covering and two Plastic Products #303 electrode plugs were used to deliver brain stimulation (Plastic Products, Roanoke, Virginia). A Kopf stereotaxic apparatus (Kopf Instruments, Tujunga, California) was used in the surgical implantation of electrodes. Electrodes were MS/303 stainless steel, 32 mm in length and 0.15 mm in diameter. Self-tapping stainless steel mounting screws 0.16 mm in length along with crainioplastic liquid and powder were used in the permanent fixing of the implanted electrodes. All electrodes, mounting screws, and cement were supplied by Plastic Products, Inc., Roanoke, Virginia.

Procedure

Subjects were randomly assigned to form two groups (n = 12 in each), an anterior stimulation group and a sham stimulation (dummy electrode) group. Following assignment to groups, animals were subjected to the surgical procedure. A second experimental group with electrodes aimed at the ventromedial hypothalamus was initially included, however, histological examination of electrode placements for this group indicated that electrodes were variable in their placement and usually posterior to the ventromedial nucleus of the hypothalamus. The variability of the placements did not permit the animals to be considered as representing a meaningful experimental condition, therefore data from this group were not considered interpretable and were not included in the analysis of results.

Surgery. Animals were anesthetized with sodium pentobarbital at a dose of 50 mg/kg. Following anesthesia, each animal was placed in a stereotaxic apparatus and the skull was exposed by an incision approximately 1 inch in length. Following the incision, the skull was trephined and electrodes were implanted bilaterally in the anterior nucleus of the hypothalamus. Stereotaxic coordinates for electrode implantation were 1.0 mm posterior of bregma, 0.7 mm lateral of the midsagittal sinus, and 7.0 mm ventral from the dural surface of the brain for the anterior group and 6.5 mm anterior to the interaural line, 1.0 mm lateral of the midsagittal sinus, and 8.0 mm ventral from the dural surface of the brain for the sham group which placed the electrode tip in the lateral hypothalamus (Sherwood & Timeras, 1970; Tyrey & Nalbandov, 1972).

Measurement procedures. Following the return of each animal to preoperative weight (7 to 10 days), subjects from each group were removed from the home cage and IgG concentrations were determined through the use of radial immunodiffusion as outlined by Mancini, Carbonara, and Heremans (1965). A 0.5-ml blood sample was collected by cutting the tail vein with a sterile scalpel blade. The blood sample was allowed to clot, centrifuged at 3500 rpm for 15 minutes in order to remove cells, and serum was collected from the clotted sample. Next, 16.6 ul of serum from each sample was pipetted into a 4-mm well cut within a 1% agarose-phosphate buffered (ph 7.2) sterile saline medium contained on a 3[inches] x 1[inch] Corning glass microscope slide (Fisher Scientific Products, Dallas, Texas). The agarose medium contained a 1:20 dilution of rat anti-IgG (Sigma Chemical Company, St. Louis, Missouri). The serum-agarose anti-IgG complex was allowed to incubate at 25 [degrees] C for 24 hours. At the end a 24-hr period, ring diameters were measured using a Bel Art caliper (Fisher Scientific Product, Dallas, Texas). Concentrations of IgG were determined for samples by comparing ring diameters with ring diameters produced by reacting known concentrations of rat IgG (Sigma Chemical Company, St. Louis, Missouri). The IgG concentrations were calculated from ring diameters through the use of a linear regression equation (Y = mx + b). The logarithm of known concentrations of IgG were correlated with the ring diameters obtained during determination of a standard curve. Resulting values of the slope (m) and intercept (b) of the line were used to determine the IgG concentrations of sample ring diameters. The antilog of y produced the predicted IgG concentration of the sample. Average linear correlations between ring diameters and known concentrations of IgG were determined to be 0.98 for all standards. The above measurement served as a baseline measure of IgG concentrations for each subject.

Brain stimulation. Following the determination of baseline concentrations of IgG, a period of 4 days elapsed before the next phase of the experiment. At the end of this period, each animal was placed in an operant chamber where intracranial brain stimulation was delivered by a Grass S48 brain stimulator through a bifurcated cable. Animals in the anterior group received one train of rectangular wave form pulses every 10 s up to a maximum of 6 V with the duration of each train lasting 1 s.

Intensity of brain stimulation was adjusted for each animal by beginning the first session at 1 V and increasing by 1-V increments every 2 minutes until either a maximum of 6 V was reached or until an aversive reaction to brain stimulation was observed. If an aversive reaction was observed, the intensity of brain stimulation was lowered in 1-V units until the reaction(s) were no longer observed and that value of intensity was held constant for each animal throughout the experiment. An aversive reaction was defined as flinching, jumping, and/or vocalization at stimulus onset. For animals in the anterior group the level of current used for brain stimulation ranged from 100 [[micro]ampere] to 400 [[micro]ampere] across the 12 animals with an average current of 191.67 [[micro]ampere] and a median current of 200 [[micro]ampere].

Animals in the dummy electrode group had the bifurcated cable attached for the same amount of time but brain stimulation was not delivered. Each train of brain stimulation delivered 100 stimulus pulses per second with the duration of each stimulus pulse lasting 0.2 msec.

Following 1 hr of brain stimulation, IgG concentrations for each animal was determined using the Mancini technique at 3, 6, 12, and 24 hours postbrain stimulation in a counterbalanced sequence. A 4-day period was interposed between each brain stimulation-IgG determination to preclude the possibility of carry-over effects.

Histology. Following the conclusion of the experiment, histological analysis of the brain was performed. The brain was removed from the skull following perfusion with 0.9% physiological saline and 10% formalin. Brain tissue was embedded in celulodin and sliced with a clinical microtome at 30 [[micro]meter]. Tissue was stained with Thionin and mounted on 3- x 1-inch Corning glass slides for microscopic examination.

Results

Results of histological examination of brain tissue indicated that electrode placements for animals in the anterior hypothalamic group were within the anterior hypothalamus. based on radial immunodiffusion, mean IgG concentrations were less for animals in the anterior group than in the control group. See Figure 1 for a graphic representation of poststimulation mean IgG concentrations following adjustment for prestimulation baseline IgG.

These data were analyzed by a 2-way analysis of covariance with repeated measures on poststimulation periods using baseline IgG as the covariate. Covariance analysis adjusts each animal's poststimulation IgG concentrations for differences in initial baseline IgG values. Mean baseline levels and standard deviations of IgG concentrations for the sham group and the anterior group were M = 7.71, SD = 23.4 and M = 7.09, SD = 2.56, respectively. A significant difference was found in overall IgG concentrations between the anterior group and sham group [F (1, 21) = 7.96, p = .0102]. Neither the poststimulation period [F (3, 63) = [less than] 1, p = .473] nor the groups by poststimulation period interaction [F (3, 63) = [less than] 1, p = .551] were significant. The lack of a significant interaction indicates that comparisons between groups at each poststimulation period are inappropriate.

Figure 2 displays the relationship between number stimulations and IgG concentration collapsed across poststimulation intervals. There appears to be an increase in IgG with number of measurements.

A subsequent analysis of covariance was performed to assess the effect of the number of measurements on IgG concentrations between the anterior group and the sham group. A significant difference was found between the anterior group and sham group across the number of measurement periods IF (1, 21) = 7.96, p = .0102]. A significant within-subjects effect was found across measurements [F (3, 63) = 4.89, p = .0040]. The number of measurements by groups interaction was not significant IF (3, 63) = [less than] 1, p = .9799] indicating that comparisons between groups at each measurement are inappropriate.

Discussion

Data from this study support the conclusion that electrical stimulation of the anterior hypothalamus reduces concentrations of IgG in rat serum. These results provide further support for the role of the anterior hypothalamus in the modulation of immunoresponsivity. In addition, the present study demonstrates that at least one of the immunoglobulins involved in the humoral immune response (IgG) can be affected through direct stimulation of anterior hypothalamic structures.

Under the parameters of the present study the anterior group displayed depressed IgG levels as compared to the sham group. Inspection of Figure 1 indicates that the anterior group displays consistently lower IgG concentrations when compared to the sham group. The IgG levels reported are adjusted for differences in baseline levels for each group through the use of covariance analysis. Order or sequential effects are controlled through the use of counterbalancing of poststimulation measurement periods. A subsequent analysis of covariance was performed to assess order or sequential effects and this analysis yielded a significant effect on the number of measurements. Inspection of Figure 2 indicates that IgG concentrations increase as a function of the number of measurements taken. Research has shown that handling mice for 2 min per day resulted in a decreased primary IgG response to antigenic challenge (Moynihan, Brenner, Koota, Breneman, Cohen, & Ader, 1990), thus simply handling and/or measuring immunoglobulins in the intact animal can have effects which may obscure experimental manipulations. The use of adequate control groups and design considerations such as counterbalancing can increase the internal validity of research in this area.

The specific mechanism(s) by which anterior hypothalamic stimulation influences IgG production is currently unknown. Lowered IgG antibody titers produced through anterior hypothalamic stimulation may result from a number of factors. Anterior hypothalamic stimulation could produce lowered IgG levels through neuroendocrine influences via the pituitary gland and or through direct connections to IgG producing lymphocytes via the autonomic nervous system (ANS). Additionally, anterior hypothalamic influences may interact with limbic system structures to modulate the production of IgG via hormonal or ANS pathways (Nance et al., 1987).

Previous studies investigating the role of the anterior hypothalamus in humoral immunity have either lesioned the anterior hypothalamus or recorded neuronal activity in the anterior area in the face of a challenge with a known antigen (Abramsky et al., 1987; Saphier et al., 1987; Tyrey & Nalbandov, 1972). The form of the response of antibody in the face of antigenic challenge is somewhat dependent on the class of antibody measured (Barrett, 1978).

IgG responsivity in the face of antigen is biphasic, and thus this antibody is sometimes referred to as the memory component of the antibody response (Barrett, 1978). Following an initial challenge with antigen, IgG levels increase over the first few days or weeks and then begin to drop. If subsequently challenged within a 20- to 30-day period, a dramatic increase in antibody levels are observed. This dramatic increase is usually noted with respect to IgG (Barrett, 1978). Nance et al. (1987) showed that lesions of the lateral septal area decreased IgG concentrations whereas hippocampal lesions increased IgG concentrations in the face of antigenic challenge.

Direct environmental events or conditioned cues to threat could modulate hypothalamic influences on IgG concentrations. Research employing Pavlovian conditioning procedures have found that a previously neutral stimulus paired with either an immunosuppressive agent or an antigen can become a signal for increases or decreases in immune responsivity (Ader & Cohen, 1981; Ader et al., 1993). The effects of stress and other psychosocial factors on immunity (O'Leary, 1990) may influence hypothalamic activity thereby compromising acquired immunity as well as affecting the ability to develop antibodies to newly encountered antigens.

A growing body of evidence links chronic stress, depression, and personality traits as possible contributors to decreases in disease resistance and that intervention strategies can produce enhanced immunocompetence in human subjects (Maier, Watkins, & Fleshner, 1994; O'Leary, 1990; Siegal et al., 1991; Zakowski et al., 1992). For example, Glaser, Pearson, Bonneau, Esterling, Atkinson, and Kiecolt-Glaser (1993) found that cell-mediated immunity to Epstein-Barr virus decreased in subjects just prior to the administration of academic exams. Thus, direct environmental stressors and conditioned signals for threat and their effects on immunity may both be mediated by hypothalamic activity as well as other areas of the limbic system.

In conclusion, the present study directly stimulated the anterior hypothalamus but did not provide a challenge with a known antigen prior to measuring IgG concentrations. Anterior hypothalamic stimulation may mimic neural activation following antigenic challenge. It is possible that anterior hypothalamic activation may produce a decrease in IgG concentrations in the absence of known antigenic stimulation. The results of the present study suggest that neural activation may modulate the existing level(s) of IgG thereby changing the current level of immunity present in the intact organism. It may be speculated that changing the current level(s) of existing antibodies may result in a change in the immune system's ability to provide protection for the organism. Future studies should continue to map the hypothalamus for the effects of stimulation on immunoglobulins and it is possible that both suppression and facilitation will be found. Studies of the effects of hypothalamic stimulation on survival rates in rats exposed to life threatening events such as exposure to carcinogenic substances would be interesting. Continued research will clarify the role of hypothalamic structures the organism's response to disease.

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Author:Lambert, Paul L.; Harrell, Ernest H.; Kelly, Kimberly
Publication:The Psychological Record
Date:Jun 22, 1998
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