Dynamics of cachexia-associated inflammatory changes in the brain accompanying intra-abdominal fibrosarcoma growth in Wistar rats.

Accumulated data indicate that inflammation affecting brain structures participates in the development of cancer-related cachexia. However, the mechanisms responsible for the induction and progression of cancer-related neuroinflammation are still not fully understood. Therefore, we studied the time-course of neuroinflammation in selected brain structures and cachexia development in tumor-bearing rats. After tumor cells inoculation, specifically on the 7th, 14th, 21st, and 28th day of tumor growth, we assessed the presence of cancer-associated cachexia in rats. Changes in gene expression of inflammatory factors were studied in selected regions of the hypothalamus, brain stem, and circumventricular organs. We showed that the initial stages of cancer growth (7th and 14th day after tumor cells inoculation), are not associated with cachexia, or increased expression of inflammatory molecules in the brain. Even when we did not detect cachexia in tumor-bearing rats by the 21st day of the experiment, the inflammatory brain reaction had already started, as we found elevated levels of interleukin 1 beta, interleukin 6, tumor necrosis factor alpha, and glial fibrillary acidic protein mRNA levels in the nucleus of the solitary tract. Furthermore, we found increased interleukin 1 beta expression in the locus coeruleus and higher allograft inflammatory factor 1 expression in the vascular organ of lamina terminalis. Ultimately, the most pronounced manifestations of tumor growth were present on the 28th day post-inoculation of tumor cells. In these animals, we detected cancer-related cachexia and significant increases in interleukin 1 beta expression in all brain areas studied. We also observed significantly decreased expression of the glial cell activation markers allograft inflammatory factor 1 and glial fibrillary acidic protein in most brain areas of cachectic rats. In addition, we showed increased expression of cluster of differentiation 163 and cyclooxygenase 2 mRNA in the hypothalamic paraventricular nucleus, A1/C1 neurons, and area postrema of cachectic rats. Our data indicate that cancer-related cachexia is associated with complex neuroinflammatory changes in the brain. These changes can be found in both hypothalamic as well as extrahypothalamic structures, while their extent and character depend on the stage of tumor growth.

Chronic propranolol treatment moderately attenuated development of N-methyl-N-nitrosourea-induced mammary carcinoma in female rats.

The sympathetic nervous system participates in the development and progression of several cancer types and this effect is mediated mainly via ß-adrenergic signaling. However, the potential of ß-adrenergic signaling blockade to prevent cancer development after exposure to carcinogens has not been investigated, yet. Therefore, in our study, we determined the effect of the ß-blocker propranolol on the development and progression of mammary cancer induced in female rats by administration of the chemical carcinogen N-methyl-N-nitrosourea (MNU). The propranolol treatment (20 mg/kg body weight) started 12 days after MNU administration and lasted 10 weeks. We found that both saline and propranolol treatment significantly increased gene expression of the catecholamine-synthesizing enzyme tyrosine hydroxylase, indicating that repeated injection of saline or propranolol-induced stress in these two groups. However, compared to the vehicle-treated group, propranolol slightly delayed the development and moderately reduced the incidence of mammary carcinoma in animals. To evaluate the mechanisms mediating the effect of propranolol on the development of MNU-induced cancer, we investigated several parameters of the tumor microenvironment and found that propranolol increased gene expression of Casp3. Our data indicate that propranolol treatment that starts after exposure to carcinogens might represent a new, useful approach for preventing the development of cancer, especially in stressed individuals. However, the potential efficiency of propranolol treatment for preventing cancer development and progression in individuals exposed to carcinogens needs further investigation.

Vagotomy Affects Lipopolysaccharide-Induced Changes of Urocortin 2 Gene Expression in the Brain and on the Periphery.

The corticotropin-releasing hormone family of peptides is involved in regulating the neuroendocrine stress response. Also, the vagus nerve plays an important role in the transmission of immune system-related signals to brain structures, thereby orchestrating the neuroendocrine stress response. Therefore, we investigated gene expression of urocortin 2 (Ucn2) and c-fos, a markers of neuronal activity, within the hypothalamic paraventricular nucleus (PVN), a brain structure involved in neuroendocrine and neuroimmune responses, as well as in the adrenal medulla and spleen in vagotomized rats exposed to immune challenge. In addition, markers of neuroendocrine stress response activity were investigated in the adrenal medulla, spleen, and plasma. Intraperitoneal administration of lipopolysaccharide (LPS) induced a significant increase of c-fos and Ucn2 gene expression in the PVN, and adrenal medulla as well as increases of plasma corticosterone levels. In addition, LPS administration induced a significant increase in the gene expression of tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) in the adrenal medulla. In the spleen, LPS administration increased gene expression of c-fos, while gene expression of TH and PNMT was significantly reduced, and gene expression of Ucn2 was not affected. Subdiaphragmatic vagotomy significantly attenuated the LPS-induced increases of gene expression of c-fos and Ucn2 in the PVN and Ucn2 in the adrenal medulla. Our data has shown that Ucn2 may be involved in regulation of the HPA axis in response to immune challenge. In addition, our findings indicate that the effect of immune challenge on gene expression of Ucn2 is mediated by vagal pathways.

Changes in gene expression in brain structures related to visceral sensation, autonomic functions, food intake, and cognition in melanoma-bearing mice.

The brain exerts complex effects on the initiation and progression of cancer in the body. However, the influence of cancer localized in peripheral tissues on the brain has been only partially described. Therefore, we investigated gene expression in brain structures that participate in transmitting viscerosensory signals, regulating autonomic functions and food intake, as well as cognition in C57Bl/6J mice with B16-F10 melanoma. In addition, we investigated the relationship between peripheral inflammation and neuroinflammation. We found increased neuronal activity in the nucleus of the solitary tract of tumor-bearing mice, whereas neuronal activity in the A1/C1 catecholaminergic cell group, parabrachial nucleus, lateral hypothalamic area, ventromedial nucleus of the hypothalamus, paraventricular nucleus of the hypothalamus, and hippocampus was decreased. In the majority of investigated brain structures, we found increased gene expression of IL-1ß, whereas gene expression of IL-6 and NF-κB was reduced or unchanged compared with controls. Melanoma-bearing mice also showed increased gene expression of tyrosine hydroxylase in the A1/C1 catecholaminergic cell group, nucleus of the solitary tract, and locus coeruleus, as well as reduced mRNA levels of hypocretin neuropeptide precursor protein in the lateral hypothalamic area, and proopiomelanocortin in the arcuate nucleus. In addition, we found reduced mRNA levels of Bcl-2, brain-derived neurotrophic factor, and doublecortin in the hippocampus. Our data indicate that skin melanoma induces complex changes in the brain, and these changes are most probably caused by cancer-related signals mediated by pro-inflammatory cytokines.

Cachexia induced by Yoshida ascites hepatoma in Wistar rats is not associated with inflammatory response in the spleen or brain.

Recent data indicate that peripheral, as well as hypothalamic pro-inflammatory cytokines play an important role in the development of cancer cachexia. However, there are only a few studies simultaneously investigating the expression of inflammatory molecules in both the periphery and hypothalamic structures in animal models of cancer cachexia. Therefore, using the Yoshida ascites hepatoma rat's model of cancer cachexia we investigated the gene expression of inflammatory markers in the spleen along with the paraventricular and arcuate nuclei, two hypothalamic structures that are involved in regulating energy balance. In addition, we investigated the effect of intracerebroventricular administration of PS-1145 dihydrochloride (an Ikß inhibitor) on the expression of selected inflammatory molecules in these hypothalamic nuclei and spleen. We observed significantly reduced food intake in tumor-bearing rats. Moreover, we found significantly decreased expression of IL-6 in the spleen as well as decreased NF-κB in the paraventricular nucleus of rats with Yoshida ascites hepatoma. Similarly, expression of TNF-α, IL-1ß, NF-κB, and COX-2 in the arcuate nucleus was significantly reduced in tumor-bearing rats. Administration of PS-1145 dihydrochloride reduced only the gene expression of COX-2 in the hypothalamus. Based on our findings, we suggest that the growing Yoshida ascites hepatoma decreased food intake by mechanical compression of the gut and therefore this model is not suitable for investigation of the inflammation-related mechanisms of cancer cachexia development.

Sympathectomy reduces tumor weight and affects expression of tumor-related genes in melanoma tissue in the mouse.

Accumulated evidence indicates that sympathetic nerves may potentiate tumor growth, including melanoma. To elucidate possible mechanisms for this effect, we performed chemical sympathectomy by intraperitoneal (i.p.) injection of the neurotoxin 6-hydroxydopamine hydrobromide (100 mg/kg of body weight); in nine adult male C57BL/6J mice; nine control mice received i.p. vehicle (VEH). Seven days later, all mice were injected subcutaneously with 3 × 10(3) B16-F10 melanoma cells. Mice were euthanized 20 d after injection of melanoma cells, for measurement of tumor weight and expression of genes related to sympathetic signaling, apoptosis, hypoxia and angiogenesis in tumor tissue. To assess potential involvement of the hypothalamo-pituitary-adrenocortical axis in the effect of sympathectomy on melanoma growth, concentrations of plasma corticosterone and level of glucocorticoid receptor mRNA in tumor tissue were determined. We found that sympathectomy significantly attenuated melanoma growth (tumor weight 0.29 ± 0.16 g versus 1.02 ± 0.30 g in controls; p < 0.05). In tumor tissue from sympathectomized mice, we found significantly increased gene expression (measured by real-time PCR), relative to VEH-injected controls, of tyrosine hydroxylase, neuropeptide Y and glucocorticoid receptor (all p < 0.05), and alpha1, beta1 and beta3 adrenergic receptors (all p < 0.025), and factors related to apoptosis (Bcl-2 and caspase-3; p < 0.05) and hypoxia (hypoxia inducible factor 1 alpha) (p = 0.005). Plasma corticosterone concentrations were significantly elevated (p < 0.05) in these mice. Our findings indicate that sympathectomy induces complex changes in the tumor microenvironment reducing melanoma growth. Such complex changes should be considered in the prediction of responses of cancer patients to interventions affecting sympathetic signaling in tumor tissue and its environment.

Subdiaphragmatic vagotomy enhances stress-induced epinephrine release in rats.

Neuroendocrine stress response is regulated by several feedback loops. Since it has been suggested that afferent vagal pathways contribute to these feedback loops, we examined the effect of surgical subdiaphragmatic vagotomy on both baseline and stress-induced increases in plasma epinephrine, norepinephrine, and corticosterone levels in vagotomized and sham-operated Sprague Dawley rats. On either the 3rd or 14th day following vagotomy, the animals were exposed to acute immobilization stress and blood from the jugular vein was collected both before and during stress exposure. We found that vagotomy significantly enhanced immobilization-induced increases of plasma epinephrine, norepinephrine, and corticosterone levels on the 3rd day following surgery. However, on the 14th day following surgery, vagotomy enhanced only increase of plasma epinephrine levels in stressed rats. Our data indicate that afferent pathways of the vagus nerve are involved in negative feedback regulation of epinephrine secretion from the adrenal medulla during stressful conditions. We hypothesize that this feedback mechanism might be mediated by the binding of circulating epinephrine on ß2-adrenergic receptors localized on sensory endings of the vagus nerve.

Ambiguous effect of signals transmitted by the vagus nerve on fibrosarcoma incidence and survival of tumor-bearing rats.

While the parasympathetic nervous system appears to be involved in the regulation of tumor progression, its exact role is still unclear. Therefore, using a rat BP6-TU2 fibrosarcoma tumor model, we investigated the effect of (1) reduction of vagal activity produced by subdiaphragmatic vagotomy; and (2) enhancement of vagal activity produced by continuous delivery of electric impulses to the cervical part of the vagus nerve on tumor development and survival of tumor-bearing rats. We also evaluated the expression of cholinergic receptors within in vitro cultivated BP6-TU2 cells. Interestingly, we found that both, vagal stimulation and subdiaphragmatic vagotomy slightly reduced tumor incidence. However, survival of tumor-bearing rats was not affected by any of the experimental approaches. Additionally, we detected mRNA expression of the α1, α2, α5, α7, and α10 subunits of nicotinic receptors and the M1, M3, M4, and M5 subtypes of muscarinic receptors within in vitro cultivated BP6-TU2 cells. Our data indicate that the role of the vagus nerve in modulation of fibrosarcoma development is ambiguous and uncertain and requires further investigation.

Regulation of nonclassical renin-angiotensin system receptor gene expression in the adrenal medulla by acute and repeated immobilization stress.

The involvement of the nonclassical renin-angiotensin system (RAS) in the adrenomedullary response to stress is unclear. Therefore, we examined basal and immobilization stress (IMO)-triggered changes in gene expression of the classical and nonclassical RAS receptors in the rat adrenal medulla, specifically the angiotensin II type 2 (AT2) and type 4 (AT4) receptors, (pro)renin receptor [(P)RR], and Mas receptor (MasR). All RAS receptors were identified, with AT2 receptor mRNA levels being the most abundant, followed by the (P)RR, AT1A receptor, AT4 receptor, and MasR. Following a single IMO, AT2 and AT4 receptor mRNA levels decreased by 90 and 50%, respectively. Their mRNA levels were also transiently decreased by repeated IMO. MasR mRNA levels displayed a 75% transient decrease as well. Conversely, (P)RR mRNA levels were increased by 50% following single or repeated IMO. Because of its abundance, the function of the (P)RR was explored in PC-12 cells. Prorenin activation of the (P)RR increased phosphorylation of extracellular signal-regulated kinase 1/2 and tyrosine hydroxylase at Ser(31), likely increasing its enzymatic activity and catecholamine biosynthesis. Together, the broad and dynamic changes in gene expression of the nonclassical RAS receptors implicate their role in the intricate response of the adrenomedullary catecholaminergic system to stress.

Chemical sympathectomy increases neutrophil-to-lymphocyte ratio in tumor-bearing rats but does not influence cancer progression.

The sympathetic nervous system regulates many immune functions and modulates the anti-tumor immune defense response, too. Therefore, we studied the effect of 6-hydroxydopamine induced sympathectomy on selected hematological parameters and inflammatory markers in rats with Yoshida AH130 ascites hepatoma. We found that chemically sympathectomized tumor-bearing rats had significantly increased neutrophil-to-lymphocyte ratio, leukocyte-to-lymphocyte ratio, and plasma levels of tumor necrosis factor alpha. Although our findings showed that sympathetic denervation in tumor-bearing rats led to increased neutrophil-to-lymphocyte ratio, that is an indicator of the disease progression, we found no significant changes in tumor growth and survival of sympathectomized tumor-bearing rats.

Bradykinin B2 receptor in the adrenal medulla of male rats and mice: glucocorticoid-dependent increase with immobilization stress.

Bradykinin, acting via the bradykinin B2 receptor (B2R), is a potent stimulator of adrenomedullary catecholamine biosynthesis and release and likely plays an important role in the adrenomedullary stress response. However, the effects of stress on the expression of this receptor in the adrenal medulla are currently unclear. Here, we examined the changes in adrenomedullary B2R gene expression in male rats in response to single (1 time) and repeated (6 times) exposure to 2 hours immobilization stress (IMO). Immediately after 1 or 6 times IMO, B2R mRNA levels were increased by 9-fold and 7-fold, respectively, and returned to unstressed control levels 3 hours later. This large, but transient, increase in mRNA elicited a doubling of protein levels 3 hours after the stress exposure. Next, the role of the hypothalamic-pituitary-adrenocortical axis in the stress-induced upregulation of B2R gene expression was examined. Treatment with endogenous (corticosterone) and synthetic (dexamethasone) glucocorticoids dose-dependently increased B2R mRNA levels in adrenomedullary-derived PC12 cells. Furthermore, cortisol supplementation at levels mimicking stress exposure elevated B2R mRNA levels in the adrenal medulla of hypophysectomized rats. In response to 1 exposure to IMO, the stress-triggered rise in plasma corticosterone and adrenomedullary B2R mRNA levels was attenuated in CRH-knockout mice and absent in pharmacologically adrenalectomized rats, indicating a requirement for glucocorticoids in the upregulation of B2R gene expression with stress. Overall, the increase in B2R gene expression in response to the stress-triggered rise in glucocorticoids likely enhances catecholamine biosynthesis and release and may serve as an adaptive response of the adrenomedullary catecholaminergic system to stress.

Stress-induced changes in gene expression of urocortin 2 and other CRH peptides in rat adrenal medulla: involvement of glucocorticoids.

The corticotropin-releasing hormone (CRH) family regulates the endocrine stress response. Here, we examined the effect of immobilization stress (IMO) on gene expression of adrenomedullary CRH family members. Urocortin 2 (Ucn2) has the highest basal gene expression and is increased by > 30-fold in response to single IMO and about 10-fold after six daily repeated IMO. IMO also induced a smaller rise in CRH (six-fold) and CRH receptor type 1 (CRHR1; two-fold with single IMO). The influence of glucocorticoids was examined. Dexamethasone (DEX) or corticosterone greatly increased Ucn2 mRNA levels in PC12 cells in a dose-dependent manner. The DEX elicited rise in Ucn2 was abolished by actinomycin D pre-treatment, indicating a transcriptionally mediated response. DEX also triggered a rise in CRHR1 and lowered CRH mRNA levels. In CRH-knockout mice, where the IMO-induced rise in corticosterone was attenuated, the response of IMO on Ucn2, as well as CRHR2 mRNAs was absent. Overall, the results suggest that the stress-triggered rise in glucocorticoids is involved in the large induction of Ucn2 mRNA levels by IMO, which may allow Ucn2 to act in an autocrine/paracrine fashion to modulate adrenomedullary function, or act as an endocrine hormone.

Regulation of angiotensin II type 2 receptor gene expression in the adrenal medulla by acute and repeated immobilization stress.

While the renin-angiotensin system is important for adrenomedullary responses to stress, the involvement of specific angiotensin II (Ang II) receptor subtypes is unclear. We examined gene expression changes of angiotensin II type 1A (AT(1A)) and type 2 (AT(2)) receptors in rat adrenal medulla in response to immobilization stress (IMO). AT(2) receptor mRNA levels decreased immediately after a single 2-h IMO. Repeated IMO also decreased AT(2) receptor mRNA levels, but the decline was more transient. AT(1A) receptor mRNA levels were unaltered with either single or repeated IMO, although binding was increased following repeated IMO. These effects of stress on Ang II receptor expression may alter catecholamine biosynthesis, as tyrosine hydroxylase and dopamine ß-hydroxylase mRNA levels in PC12 cells are decreased with Ang II treatment in the presence of ZD7155 (AT(1) receptor antagonist) or with CGP42112 (AT(2) receptor agonist) treatment. Involvement of stress-triggered activation of the hypothalamic-pituitary-adrenocortical or sympathoadrenal axis in AT(2) receptor downregulation was examined. Cultured cells treated with the synthetic glucocorticoid dexamethasone displayed a transcriptionally mediated decrease in AT(2) receptor mRNA levels. However, glucocorticoids are not required for the immediate stress-triggered decrease in AT(2) receptor gene expression, as demonstrated in corticotropin-releasing hormone knockout (Crh KO) mice and hypophysectomized rats, although they can regulate basal gene expression. cAMP and pituitary adenylate cyclase-activating polypeptide also reduced AT(2) receptor gene expression and may mediate this response. Overall, the effects of stress on adrenomedullary AT(1A) and AT(2) receptor expression may contribute to allostatic changes, such as regulation of catecholamine biosynthesis.

Stress triggered changes in expression of genes for neurosecretory granules in adrenal medulla.

With acute stress, the release of adrenomedullary catecholamines is important for handling the emergency situation. However, when chronic or repeated, stress alters the allostatic load and leads to a hyperadrenergic state, resulting in the development or worsening of a wide range of diseases. To help elucidate the mechanism, we examined the effects of single and repeated immobilization stress on gene expression of components of neurosecretory vesicles in the adrenal medulla. Male Sprague-Dawley rats were exposed to immobilization stress once for 2 h (1× IMO) or daily for six consecutive days (6× IMO). Compared to unstressed controls, 1× IMO elevated gene expression of vesicular monoamine transporter 2 (VMAT2). In response to 6× IMO, not only was VMAT2 mRNA still elevated, but chromogranin A (CgA) and chromogranin B (CgB) mRNAs were also increased two to three-fold above basal levels. To investigate the possible role of the hypothalamic-pituitary-adrenal axis in the induction of VMAT2, PC12 cells were treated with the synthetic glucocorticoid dexamethasone, which was found to elevate VMAT2 mRNA expression. The findings suggest that following repeated stress, elevations of various components of neurosecretory vesicles in the adrenal can facilitate more efficient utilization of the well-characterized heightened catecholamine levels.