Pyridostigmine bromide exposure creates chronic, underlying neuroimmune disruption in the gastrointestinal tract and brain that alters responses to palmitoylethanolamide in a mouse model of Gulf War Illness.

Gulf War Illness (GWI) is a chronic multisymptom illness that includes gastrointestinal disorders. Although the exact etiology of GWI is unknown, exposure to the drug pyridostigmine bromide (PB) is considered a major factor. Exposure to PB drives enteric neuroinflammation, promotes immunosuppression, and alters physiological functions of the colon in the short term but whether exposure to PB is sufficient to promote long term dysfunction is not known. Here, we tested whether exposure to PB is sufficient to drive long term changes that reflect GWI, and whether the endogenous anti-inflammatory mediator palmitoylethanolamide (PEA) is sufficient to reduce the detrimental effects of PB in the gut and brain of mice. Exposure to PB alone was not sufficient to cause major changes in neuromuscular transmission but did drive major changes by altering the effects of PEA. Calcium imaging data show that the mechanisms responsible include a shift in receptor signaling mediated by TRPV1, endocannabinoids, and peroxisome proliferator-activated receptors alpha (PPARα). Additional mechanisms include the development of glial reactivity and changes in enteric neurochemical coding and survival. PB and PEA caused major shifts in pro-inflammatory cytokines/chemokines in the brain and colon that persisted up to 5 months following exposure. Many of the effects of PB and PEA exhibit significant sex differences. Together, these results highlight novel mechanisms whereby PB promotes long-lasting changes in nervous system and immune function by inducing occult neuroplasticity that is revealed by subsequent exposure to unrelated drugs in a sex dependent manner.

Influence of Stress on the Vitamin D-Vitamin D Receptor System, Macrophages, and the Local Inflammatory Milieu in Endometriosis.

We previously demonstrated the negative impact of stress in an animal model of endometriosis. Although its role is unclear, altered levels of vitamin D (VitD) have been found in patients with this condition. VitD signaling through the VitD receptor (VDR) has anti-proliferative properties and induces an anti-inflammatory phenotype in macrophages. We hypothesized that stress impacts the vitamin D-VDR system, influencing macrophage behavior and the local inflammatory milieu in endometriosis. Endometriosis was surgically induced in female Sprague-Dawley rats, which were then exposed to uncontrollable, controllable, or no stress for 10 days. Sham controls received sutures only. VitD levels were measured by ELISA; cytokine levels by multiplex assay and PCR; and VDR expression and macrophage numbers assessed by immunohistochemistry and immunofluorescence. VDR expression in patient samples was assessed by immunohistochemical staining of a tissue microarray. Serum VitD levels were higher in endometriosis animals compared with sham (p < 0.01) with no significant effect of stress. Uncontrollable stress increased macrophage infiltration (p < 0.01) and VDR expression in vesicles, which were attenuated by controllable stress. Macrophage infiltration correlated with vesicle area (p < 0.05), and peritoneal vitamin D levels correlated with vesicle VDR expression (r = 0.81, p < 0.01). Decreased expression of chemokine ligand 2 (p < 0.05) and TGFß was observed in endometriosis with uncontrollable stress, whereas IL12 increased with controllable stress. Differential expression of VDR was observed in patient tissues. Stress exacerbates development of cysts in endometriosis through mechanisms that include macrophage recruitment, cytokine changes, and a potentially perturbed VitD:VDR axis, suggesting an impact on the local inflammatory environment.

Gastrointestinal neuroimmune disruption in a mouse model of Gulf War illness.

Gulf War illness (GWI) is a chronic multisymptom disorder that is prominent in Gulf War veterans. Major unexplained symptoms of GWI include functional gastrointestinal disorders and undiagnosed illnesses, including neurologic disorders. Exposure to the antinerve gas drug pyridostigmine bromide (PB) is linked to the development of GWI, but the exact mechanisms remain unclear. Here, we tested the hypothesis that PB alters gut function by disrupting the neural and immune systems of the intestine. We exposed male and female mice to physiologically comparable amounts of PB that match the dose, route, and time frame of exposure experienced by Gulf War veterans and assessed the acute and chronic impacts on gastrointestinal functions, the functional architecture of the enteric nervous system, and immune responses in the gut and brain. Exposure to PB drove acute alterations to colonic motility and structure in both male and female mice that transitioned to chronic changes in gut functions. PB drove acute alterations to enteric neural and glial activity, glial reactivity, and neuron survival with glial reactivity persisting into the chronic phase in male mice. Despite having no effect on colonic permeability, exposure to PB caused major shifts in the expression of proinflammatory cytokines and chemokines in the colon and brain that suggest immunosuppressive effects. Interestingly, immune disruption was still evident in the colon and brain in female animals at 1 mo following exposure to PB. Together, our results show that the paradigm of PB exposure experienced by veterans of the Persian Gulf War contributes to long-lasting pathophysiology by driving enteric neuroinflammation, promoting immunosuppression, and altering functional anatomy of the colon in a sex-dependent manner.-Hernandez, S., Fried, D. E., Grubisic, V., McClain, J. L., Gulbransen, B. D. Gastrointestinal neuroimmune disruption in a mouse model of Gulf War illness.

Impact of Psychological Stress on Pain Perception in an Animal Model of Endometriosis.

PURPOSE: Pain in patients with endometriosis is considered a significant source of stress but does not always correlate with severity of the condition. We have demonstrated that stress can worsen endometriosis in an animal model. Here, we tested the impact of a psychological stress protocol on pain thresholds and pain receptors. METHODS: Endometriosis was induced in female rats by suturing uterine horn tissue next to the intestinal mesentery. Sham rats had sutures only. Rats were exposed to water avoidance stress for 7 consecutive days or handled for 5 minutes (no stress). Fecal pellets and serum corticosterone (CORT) levels were measured as an index of anxiety. Pain perception was assessed using hot plate and Von Frey tests. Substance P, enkephalin, endomorphin-2, Mu opioid receptor (MOR), and neurokinin-1 receptor expression in the spinal cord were measured by immunohistochemistry. RESULTS: Fecal pellets and CORT were significantly higher in the endo-stress (ES) group than endo-no stress (ENS; P < .01) and sham-no stress groups (SNS; P < .01). The ES rats had more colonic damage ( P < .001 vs SNS; P < .05 vs ENS), vesicle mast cell infiltration ( P < .01 vs ENS), and more severe vesicles than ENS. The ES developed significant hyperalgesia ( P < .05) but stress reversed the allodynic effect caused by endo ( P < .001). The MOR expression was significantly reduced in ENS versus SNS ( P < .05) and more enkephalin expression was found in endo groups. CONCLUSION: Animals subjected to stress develop more severe symptoms but interestingly stress seems to have beneficial effects on abdominal allodynia, which could be a consequence of the stress-induced analgesia phenomenon.

Endometriosis Is Associated With a Shift in MU Opioid and NMDA Receptor Expression in the Brain Periaqueductal Gray.

Studies have examined how endometriosis interacts with the nervous system, but little attention has been paid to opioidergic systems, which are relevant to pain signaling. We used the autotransplantation rat model of endometriosis and allowed to progress for 60 days. The brain was collected and examined for changes in endogenous opioid peptides, mu opioid receptors (MORs), and the N-methyl-d-aspartate subunit receptor (NR1) in the periaqueductal gray (PAG), since both of these receptors can regulate PAG activity. No changes in endogenous opioid peptides in met- and leu-enkephalin or ß-endorphin levels were observed within the PAG. However, MOR immunoreactivity was significantly decreased in the ventral PAG in the endometriosis group. Endometriosis reduced by 20% the number of neuronal profiles expressing MOR and reduced by 40% the NR1 profiles. Our results suggest that endometriosis is associated with subtle variations in opioidergic and glutamatergic activity within the PAG, which may have implications for pain processing.

Double immunofluorescent staining of rat macrophages in formalin-fixed paraffin-embedded tissue using two monoclonal mouse antibodies.

The conventional approach of double immunostaining to visualize more than one protein in tissues or cells using antibodies from two different host species is not always feasible due to limitations with antibody availability. Previously reported methodologies for performing multiple immunostains on the same tissue or cells with antibodies originating from the same species are varied in their complexity, sensitivity, and approach to prevent unwanted interactions between antibodies. In the ever-expanding field of macrophage biology, much more is known about mouse and human macrophages than their rat counterparts. The limited availability of validated and well-characterized monoclonal antibodies from different species is one factor responsible for preventing advances in rat macrophage biology. Here we describe an immunostaining method for identifying and examining rat macrophages that is sufficiently sensitive for use in formalin-fixed paraffin-embedded tissue and that uses only commercially available reagents and antibodies. This method can be used to help characterize both physiological and pathophysiological processes in rat macrophages and can be adapted for use with any two antibodies from the same species of origin as long as one of the antibodies is biotinylated.

Impact of physical activity on pain perception in an animal model of endometriosis.

BACKGROUND: Symptoms of endometriosis, such as pain and infertility, are considered significant sources of stress. In many chronic conditions, exercise can act as a stress buffer and influence pain perception. We tested the impact of swimming exercise on pain perception and pain receptors in an animal model of endometriosis. METHODS: Endometriosis (Endo) was induced in female rats by suturing uterine horn tissue next to the intestinal mesentery. Sham rats received sutures only. Rats were exposed to swimming exercise for 7 consecutive days, while no-exercise rats were left in the home cage. Fecal pellets were counted after swimming as an index of anxiety, and serum corticosterone levels measured. Pain perception was assessed using the hot plate test for hyperalgesia and Von Frey test for allodynia. Mu-opioid receptor (MOR) and neurokinin-1 receptor expression in the spinal cord was measured by immunofluorescence. RESULTS: Fecal pellet counts were higher in those animals that swam (p<0.05), but no significant difference in corticosterone was found. Although Endo-exercise rats had higher colonic damage (p<0.05) with more cellular infiltration, the lesions were smaller than in Endo-no exercise rats (p<0.05). Exercise did not ameliorate the hyperalgesia, whereas it improved allodynia in both groups. MOR expression was significantly higher in Endo-exercise vs. Endo-no exercise rats (p<0.01), similar to Sham-no exercise levels. CONCLUSIONS: Our results point toward beneficial effects of swimming exercise during endometriosis progression. Physical interventions might be investigated further for their ability to reduce perceived stress and improve outcomes in endometriosis.

Stress management affects outcomes in the pathophysiology of an endometriosis model.

We have previously shown detrimental effects of stress in an animal model of endometriosis. We now investigated whether the ability to control stress can affect disease parameters. Endometriosis was surgically induced in female Sprague-Dawley rats before exposing animals to a controllable (submerged platform) or uncontrollable (no platform) swim stress protocol. Corticosterone levels and fecal pellet numbers were measured as an indicator of stress. Uncontrollable stress increased the number and size of the endometriotic cysts. Rats receiving uncontrollable stress had higher anxiety than those exposed to controllable stress or no stress and higher corticosterone levels. Uncontrollable stressed rats had more colonic damage and uterine cell infiltration compared to no stress, while controllable stress rats showed less of an effect. Uncontrollable stress also increased both colonic and uterine motility. In summary, the level of stress controllability appears to modulate the behavior and pathophysiology of endometriosis and offers evidence for evaluating therapeutic interventions.

The Probiotic Mixture VSL#3 Alters the Morphology and Secretion Profile of Both Polarized and Unpolarized Human Macrophages in a Polarization-Dependent Manner.

BACKGROUND: Patients with Inflammatory Bowel Disease (IBD), most commonly Crohn's disease (CD) or ulcerative colitis (UC), suffer from chronic intestinal inflammation of unknown etiology. Increased proinflammatory macrophages (M1) have been documented in tissue from patients with CD. Anti-inflammatory macrophages (M2) may play a role in UC given the preponderance of Th2 cytokines in this variant of IBD. Animal and clinical studies have shown that the probiotic VSL#3 can ameliorate signs and symptoms of IBD. Although animal data suggests a modulatory effect on macrophage phenotype, the effect of VSL#3 on human macrophages remains unknown. OBJECTIVE: To determine the effect of the probiotic VSL#3 on the phenotype of polarized (M1/M2) and unpolarized (MΦ) human macrophages. METHODS: Human monocyte-derived macrophages, generated by culturing monocytes with M-CSF, were left unpolarized or were polarized towards an M1 or an M2 phenotype by culture with LPS and IFN-γ or IL-4, respectively, and were then cultured in the presence or absence of VSL#3 for 3 days. Changes in macrophage morphology were assessed. Cytokine and chemokine levels in supernatants were determined by multiplex assay. RESULTS: VSL#3 decreased the granuloma-like aggregates of M1 macrophages, increased fibroblast-like M2 macrophages, and decreased fibroblast-like MΦ macrophages. VSL#3 increased the secretion of IL-1ß, IL-6, IL-10, and G-CSF by M1, M2, and MΦ macrophages. VSL#3 exposure maintained the proinflammatory phenotype of M1 macrophages, sustaining IL-12 secretion, increasing IL-23 secretion, and decreasing MDC secretion. Both VSL#3-treated M2 and MΦ macrophages secreted higher levels of anti-inflammatory and pro-healing factors such as IL-1Ra, IL-13, EGF, FGF-2, TGF-α, and VEGF, as well as proinflammatory cytokines, including IL-12 and TNF-α. CONCLUSION: Under our experimental conditions VSL#3 induced a mixed proinflammatory and anti-inflammatory phenotype in polarized and unpolarized macrophages. This differential effect could explain why patients with CD do not respond to probiotic therapy as well as patients with UC.