Adrenergic agents are prescribed for prostate cancer to facilitate urination by relaxing smooth muscle of the urethra, but it is not clear whether adrenergic therapy also affects prostate tumor growth. Sympathetic nervous system (SNS) could affect prostate tumor growth via dense innervation of the prostate gland. The purpose of this study was to (1) examine whether adrenergic agonists that selectively bind to β2- or α1- AR directly affect in vitro growth of the human prostate cancer cell line (LNCaP) and the RM-9 murine prostate cancer cell line (derived from C57Bl/6 mice), and (2) examine sympathetic innervation of host prostate and orthotopic RM-9 tumors in syngeneic C57Bl/6 mice. LNCaP and RM-9 cells were incubated for 6 or 24 hr with varying concentrations of a β2-AR (terbutaline) or an α1-AR agonist (phenylephrine) with or without a β-AR (propranolol) or an α1-AR antagonist (prazosin), respectively. Viability was assessed by MTS analysis and growth by cell cycle analysis by flow cytometry. RM-9 cells (5,000 cells) were orthotopically inoculated into the dorsolateral prostate gland of young male C57Bl/6 mice, killed 14 days later, and prostate tumors prepared for fluorescence histochemistry for localizing catecholamines or immunofluorescence for tyrosine hydroxylase. We report that phenylephrine or terbutaline increased cell viability in a dose-dependent manner at 6 hr. Incubation with 100 nM terbutaline reduced the percentage on cells in the S and G2/M phase of the RM-9 cell cycle (the replicating phase) at 24 hr, which was blocked by propranolol. Incubation with 100 nM phenylephrine had no effect on the percentage of replicating RM-9 cells at 6 hr, while the addition of the antagonist reduced RM-9 cell replication at 24 hr, possibly by a nonreceptor-mediated mechanism. Incubation with 100 nM terbutaline or phenylephrine increased the percentage on cells in the S and G2/M phase of the LNCaP cell cycle at 24 hr, which was blocked in both treatments by the addition of the appropriate antagonist. RM-9 cells preincubated with terbutaline for 24 hr, significantly increased basal levels of cAMP production compared with controls, which was blocked by the addition of the antagonist, indicating β -AR activation in vitro. Histochemical staining revealed noradrenergic nerves in close proximity to RM-9 tumor cells and TH-positive neuroendocrine cells within the tumor. These data are consistent with previous reports of AR expression on human and rat prostate cell lines, and support SNS and neuroendocrine cell modulation of RM-9 cell growth and proliferation in vivo.

Products of immune cells can communicate with the CNS affecting neural activity, neurotransmitter synthesis and release, and behavior. In rheumatoid arthritis (RA), longterm immune activation may contribute to psychological disorders that occur with higher frequency than the general population. Few studies have examined pathophysiological changes in the CNS with RA or animal models for RA. In this study, we examined the effect of immunization with complete Freund''''s adjuvant (CFA) to induce arthritis in male Lewis rats on neural activity in the brain. Rats were immunized with 0.03 mg of heat-killed Mycobacterium butyricum suspended in 100 ml of sterile endotoxin-free mineral oil or 0.9% sterile, endotoxin-free saline. Vehicle-treated and untreated animals served as controls. On day 28 post-immunization, animals were deeply anesthetized and perfused with 4% paraformaldehyde plus picric acid. Brains were dissected, prepared for immunocytochemistry and regions of the hippocampus, CA1, CA2, CA3 and the dentate DG were examined for: (1) c-fos, a neuronal activation marker or (2) tyrosine hydroxylase (TH) the rate-limiting enzyme for the synthesis of norepinephrine. In addition, the hippocampi from non-perfused brains were microdissected and analyzed for the presence of norepinephrine using high-performance liquid chromatography (HPLC). We report here an increase in c-Fos activation in pyramidal cells of the hippocampus and an increase in the density of noradrenergic fibers in all four areas of the hippocampus of arthritic rats compared with nonarthritic controls. Furthermore, the concentration of norepinephrine in the hippocampi from arthritic animals was significantly higher than in nonarthritic controls (p<0.05). Although, the significance of AP-1 transcription factors, like c-Fos protein, remains elusive, AP-1 is thought to activate various neurotransmitter systems, such as serotonin, opioid peptides, catecholamines, acetylcholine and gamma-amino butyric acid (GABA). These findings suggest widespread and prolonged activation of the hippocampus after induction of arthritis that may be contributory to disease-related depression, altered pain perception, and/or stress-related

Approximately 30,000 men die from prostate cancer every year. This type of cancer effects middle-aged and elderly men. RM-9 cells have been developed to create a mouse model to study this disease and find more effective therapies. Empirical observations, as early as the 1950s, demonstrate the deleterious effects of stress on various types of cancer. In this study, RM-9 cells were implanted in the prostate of 2-month- and 18-month-old C57Bl/6 wildtype mice, which were then restraint-stressed to measure the effects this would have on tumor development and response. Mice also underwent a battery of behavioral tests to assess their stress response. We saw stress-mediated effects on the disease progression. There was an effect of age and stress on tumor growth. Tumor growth was greater in old mice than in young, and restraint stress enhanced tumor growth regardless of age, but this effect was greater in old mice. Restraint stress affected behavior and several immune parameters. Behavior tests demonstrate: a) older non-stressed mice were less active overall compared with older stressed and younger mice; b) older mice showed a greater tendency for learned helplessness (tail-suspension test) compared with younger mice; c) stressed older mice demonstrated greater anxiety-like behavior (elevated zero maze) compared with the other groups; and d) stressed mice never regained their pre-surgical weight while non-stressed mice did. Age and stress affected circulating peripheral blood and splenic leukocyte concentrations, suggesting a possible contributory mechanism for stress-mediated changes in tumor growth.

Previous research in our laboratory showed an age-related decline in noradrenergic (NA) sympathetic innervation of spleens from male Fischer 344 (F344) rats. Since, nerve growth factor (NGF) plays an important role in determining the density of sympathetic innervation of target tissues throughout life, dysregulation of NGF expression in splenic target cells might explain the loss of sympathetic nerves. Moreover, chronically heightened sympathetic activity can also destroy nerve terminals by increased oxidative stress resulting from the breakdown of norepinephrine. The purpose of this study was to investigate whether these mechanisms are responsible for the age-related loss of splenic sympathetic nerves.  Western blot analysis of NGF protein expression was evaluated in splenic lysates from untreated young (3 month) and old (18 month) male F344 rats, and from 15-month-old rats treated with vehicle or rilmenidine [0.5 mg/kg and 1.5 mg/kg, b.i.d. (i.p.)] for 3 months to downregulation of sympathetic activity. Our studies showed a 40% decrease in NGF protein levels in spleens from old rats compared with young adults. Noteworthy, rilmenidine induced a dose-dependent effect on NGF protein levels: administration of low and high doses of rilmenidine increased splenic NGF levels by 60 and 50%, respectively, compared to vehicle group (sterile physiological saline).  Splenic NGF levels in the low-dose rilmenidine-treated group (500 µg/kg) were not different from 3-month-old F344 rats. Collectively, these findings support our hypothesis that changes in NGF protein expression might explain, at least in part, the age-related decline in sympathetic innervation of the spleen. Furthermore our date suggests that age-related changes in sympathetic activity in the spleen during middle age may precipitate the loss of NGF protein in spleens from old F344 male rats. This research is supported by NIH grant NS44032.  

Previous research in our laboratory demonstrated an age-related decline of noradrenergic (NA) sympathetic innervation in the spleens of male Fisher 344 (F344) rats, evident by 17 months of age that was not observed in 24-month-old male Brown Norway (BN) rats. Since, the growth and maintenance of mature sympathetic neurons is dependent on the synthesis of neurotrophic factors by a variety of cell types in target organs, we hypothesized that altered neurotrophic concentrations in spleens from aged F344 rats, but not in BN rats, may account for the strain-dependent differences. To test this hypothesis, total and free nerve growth factor (NGF) and neurotrophin-3 (NT-3) concentrations in spleens from young (3-month-old) and old male F344 (24-month-old) and BN (27-month-old) rats were determined by ELISA and correlated with splenic norepinephrine concentrations. Data were expressed as means + SEM, with subsequent Student’s t-test performed to determine age-related difference (p<0.05). In young adult F344 and BN rats the mean splenic concentrations of NT-3 and NGF were comparable to levels reported by other investigators. There was no effect of age on total splenic NGF concentrations (unbound + receptor bound) in either rat strain (p>0.05).    Unbound NGF concentrations in spleens from old F344 rats, and the ratio of free/bound NGF were significantly lower (p<0.05) compared with young F344 controls. There was an age-related trend toward lower free NGF content in spleens from BN rats, but this was not statistically significant (p>0.05). Conversely, the concentrations of free NT-3 in spleens from old F344 and BN rats were higher compared with young rats (p<0.05). Age-associated changes in splenic neurotrophin content correlated with changes in splenic norepinephrine content. Collectively, these findings indicate altered neurotrophin expression in the aged spleen that may contribute to the previously reported strain-related differences in the density of sympathetic nerves in lymphoid compartments of the spleen with advancing age.