Brainstem cholinergic pathways diminish cardiovascular and neuroinflammatory actions of endotoxemia in rats: Role of NFκB/α7/α4ß2AChRs signaling.

Nicotine improves endotoxic manifestations of hypotension and cardiac autonomic dysfunction in rats. Here, we test the hypothesis that brainstem antiinflammatory pathways of α7/α4ß2 nicotinic acetylcholine receptors (nAChRs) modulate endotoxic cardiovascular derangements. Pharmacologic and molecular studies were performed to determine the influence of nicotine or selective α7/α4ß2-nAChR ligands on cardiovascular derangements and brainstem neuroinflammation caused by endotoxemia in conscious rats. The i.v. administration of nicotine (50 µg/kg) abolished the lipopolysaccharide (LPS, 10 mg/kg i.v.)-evoked: (i) falls in blood pressure and spectral measure of cardiac sympathovagal balance (ratio of the low-frequency to high-frequency power, LF/HF), (ii) elevations in immunohistochemical protein expressions of NFκB and α4ß2-nAChR in medullary neurons of the nucleus tractus solitarius (NTS) and rostral ventrolateral medulla (RVLM), and (iii) decreases in medullary α7-nAChR protein expression. These favorable nicotine influences were replicated in rats treated intracisternally (i.c.) with PHA-543613 (selective α7-nAChR agonist) or 5-iodo-A-85380 (5IA, selective α4ß2-nAChRs agonist). Measurement of arterial baroreflex activity by the vasoactive method revealed that nicotine, PHA, or 5IA reversed the LPS depression of reflex bradycardic, but not tachycardic, activity. Moreover, the counteraction by nicotine of LPS hypotension was mostly inhibited after treatment with i.c. methyllycaconitine (MLA, α7-nAChR antagonist) in contrast to a smaller effect for dihydro-ß-erythroidine (DHßE, α4ß2-nAChR antagonist), whereas the associated increases in LF/HF ratio remained unaltered. The data signifies the importance of brainstem α7, and to a lesser extent α4ß2, receptors in the nicotine counteraction of detrimental cardiovascular and neuroinflammatory consequences of endotoxemia.

Thiamine preserves mitochondrial function in a rat model of traumatic brain injury, preventing inactivation of the 2-oxoglutarate dehydrogenase complex.

BACKGROUND AND PURPOSE: Based on the fact that traumatic brain injury is associated with mitochondrial dysfunction we aimed at localization of mitochondrial defect and attempted to correct it by thiamine. EXPERIMENTAL APPROACH: Interventional controlled experimental animal study was used. Adult male Sprague-Dawley rats were subjected to lateral fluid percussion traumatic brain injury. Thiamine was administered 1 h prior to trauma; cortex was extracted for analysis 4 h and 3 d after trauma. KEY RESULTS: Increased expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor receptor 1 (TNF-R1) by 4 h was accompanied by a decrease in mitochondrial respiration with glutamate but neither with pyruvate nor succinate. Assays of TCA cycle flux-limiting 2-oxoglutarate dehydrogenase complex (OGDHC) and functionally linked enzymes (glutamate dehydrogenase, glutamine synthetase, pyruvate dehydrogenase, malate dehydrogenase and malic enzyme) indicated that only OGDHC activity was decreased. Application of the OGDHC coenzyme precursor thiamine rescued the activity of OGDHC and restored mitochondrial respiration. These effects were not mediated by changes in the expression of the OGDHC sub-units (E1k and E3), suggesting post-translational mechanism of thiamine effects. By the third day after TBI, thiamine treatment also decreased expression of TNF-R1. Specific markers of unfolded protein response did not change in response to thiamine. CONCLUSION AND IMPLICATIONS: Our data point to OGDHC as a major site of damage in mitochondria upon traumatic brain injury, which is associated with neuroinflammation and can be corrected by thiamine. Further studies are required to evaluate the pathological impact of these findings in clinical settings.

Neurotropin inhibits neuroinflammation via suppressing NF-κB and MAPKs signaling pathways in lipopolysaccharide-stimulated BV2 cells.

Neurotropin (NTP) is a widely used drug in China and Japan mainly for the treatment of chronic pain and peripheral inflammation. Nevertheless, the effects of NTP on neuroinflammation have not been explored. In this study, we investigated the anti-inflammatory effects of NTP in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells and its underlying mechanisms. BV-2 cells were pretreated with NTP for 12 h before exposure to LPS. The expression of pro-inflammatory cytokines (TNF-α and IL-6) were detected by RT-PCR and EILSA at mRNA and protein levels, respectively. Western blotting was conducted to measure the protein levels of major genes in MAPKs and NF-κB signaling pathways. Results demonstrated that NTP could attenuate the production of pro-inflammatory cytokines. Furthermore, NTP inhibited the activation of NF-κB signaling by decreasing the translocation of NF-κB p65 to the nucleus and suppressed the MAPKs signaling pathway via inhibition of the phosphorylation of p38, ERK and JNK. Taken together, these findings suggest that neurotropin exerts anti-inflammatory effects by suppressing the production of pro-inflammatory mediators via inhibition of NF-κB and MAPKs signaling pathways in LPS-stimulated BV-2 cells.

Fish oil supplementation attenuates neuroinflammation and alleviates depressive-like behavior in rats submitted to repeated lipopolysaccharide.

PURPOSE: Depression is frequently associated with inflammation, whereas omega-3 polyunsaturated fatty acids (PUFAs) primarily found in fish oil possess anti-inflammatory properties. Although converging studies suggest an antidepressant effect of PUFAs, there is limited evidence directly linking the neuro-immune modulating features of PUFAs to the antidepressant actions. METHODS: Therefore, we assessed the effects of fish oil (FO) supplementation on behavioral changes, inflammatory cytokine expression and oxidative reactions in frontal cortex and hippocampus of rats following repeated peripheral immune challenge by lipopolysaccharide (LPS) for 2 weeks (500 µg/kg every other day). RESULTS: Repeated LPS administration induced the rats to a depressive-like state and increased mRNA expression of pro-inflammatory cytokines, including 1L-1ß, 1L-6 and TNF-α, in frontal cortex and hippocampus. FO supplementation attenuated the LPS-induced abnormal behavior and brain inflammatory response. Concurrent with the antidepressant action, FO also reduced LPS-induced oxidative reactions and neural apoptosis in the rat brain, as evidenced by decreased malondialdehyde (MDA) production, increased catalase activities and inhibited pro-apoptotic protein Bax mRNA expression. In addition, FO inhibited activation of NF-κB and iNOS induced by LPS. Interestingly, we found FO suppressed the activation of the inflammasome NLRP3 and ionotropic purinergic receptor P2X7R evoked by LPS, suggesting a potential anti-inflammatory mechanism for PUFAs. Besides, FO also restored the LPS-induced neurochemical disturbance, especially the balance between serotonin and kynurenine branches of tryptophan metabolism, which is tightly associated with depression. CONCLUSIONS: These findings provide novel insights into the antidepressant action of PUFAs and further strengthen the link between inflammation and depression.

Neuroinflammatory reactions in sickness behavior induced by bacterial infection: Protective effect of minocycline.

The neurological changes elicited by bacterial infection are called sickness behavior. Minocycline (MIN) is neuroprotective with a remarkable brain tissue penetration. MIN was orally administered at a dose 90 mg/kg for 3 days, whereas Escherichia coli was given as a single intraperitoneal injection (0.2 mL of 24 h growth) on the third day. After 24 h of bacterial infection, behavioral tests namely open field and forced swimming were carried out, then animals were decapitated. Rats infected with E. coli displayed reduced struggling time in forced swimming test, as well as, exploration and locomotion in open field test with reduction in neurotransmitters (norepinephrine, dopamine, and serotonin) versus elevation in the inflammatory (tumor necrosis factor-alpha, interferon-gamma) and oxidative stress (thiobarbituric acid reactive substance, reduced glutathione) biomarkers. Inflammatory infiltrates of nuclear cells were observed in brains of infected rats. MIN administration prevented the deleterious effects of E. coli infection, thus protects against sickness behavior possibly via defending from neuroinflammation.

Sulforaphane reduces lipopolysaccharide-induced proinflammatory markers in hippocampus and liver but does not improve sickness behavior.

OBJECTIVES: Acute peripheral infection is associated with central and peripheral inflammation, increased oxidative stress, and adaptive sickness behaviors. Sulforaphane (SFN) activates the transcription factor nuclear factor E2-related factor 2 (Nrf2), which upregulates antioxidant genes and lowers inflammation. The objectives of this study were to examine the effects of SFN on proinflammatory markers and Nrf2 target genes in hippocampus and liver of mice challenged with lipopolysaccharide (LPS), and to evaluate sickness response following the LPS immune challenge. METHODS: Adult Balb/c mice received SFN (50 mg/kg, i.p.) for 3 days before being injected i.p. with LPS (1 µg) to mimic an acute peripheral infection. Sickness behaviors were measured at baseline and 6 hours after LPS. Expression of proinflammatory mediators and antioxidant genes were analyzed in hippocampus and liver 6 hours after LPS. RESULTS: SFN elevated Nrf2 target genes and reduced expression of proinflammatory mediators in hippocampus and liver, but did not improve LPS-induced sickness response. DISCUSSION: The nutritional bioactive SFN displays potent anti-inflammatory properties against LPS-induced inflammation in vitro, but has not been previously assessed in vivo during peripheral infection as a potential treatment for sickness behavior. These data indicate that SFN has anti-inflammatory effects in both brain and periphery, but that longer exposure to SFN may be necessary to reduce sickness behavior.

Berberine alleviates postoperative cognitive dysfunction by suppressing neuroinflammation in aged mice.

Postoperative cognitive dysfunction (POCD) is a significant cause of morbidity after surgery, especially for the elderly. Accumulating evidence has demonstrated that neuroinflammation plays a key role in the pathogenesis of POCD. Thus, we hypothesized that berberine, an isoquinoline alkaloid with anti-inflammatory effects, could improve surgery-induced cognitive impairment. Twenty-month-old male C57BL/6 mice were subjected to exploratory laparotomy with isoflurane anesthesia to mimic the clinical human abdominal surgery. For the interventional studies, mice received berberine (10mg/kg) or vehicle intraperitoneally. For the in vitro study, we examined the effects of berberine on lipopolysaccharide (LPS)-induced inflammatory mediators by cultured BV2 cells. Behavioral tests, expressions of IBA1, tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and IL-6 were performed at the indicated time points. In the present study, we showed that surgery impaired the contextual fear memory, as evidenced by the significantly decreased freezing time to the context. This behavioral change coincided with marked increases in IBA1, TNF-α, IL-1ß, and IL-6 in the prefrontal cortex and hippocampus only at 24h but not 7 d after surgery. In BV2 cells, LPS induced significantly increased TNF-α and IL-1ß expressions. Notably, berberine treatment rescued surgery-induced cognitive impairment and inhibited the release of IBA1, IL-1ß, and IL-6 in the hippocampus. In line with the in vivo study, berberine treatment suppressed LPS-stimulated production of TNF-α and IL-1ß in BV2 cells. In conclusion, our study suggests that berberine could alleviate POCD by suppressing neuroinflammation in aged mice.

Mast cell activation contributes to sickle cell pathobiology and pain in mice.

Sickle cell anemia (SCA) is an inherited disorder associated with severe lifelong pain and significant morbidity. The mechanisms of pain in SCA remain poorly understood. We show that mast cell activation/degranulation contributes to sickle pain pathophysiology by promoting neurogenic inflammation and nociceptor activation via the release of substance P in the skin and dorsal root ganglion. Mast cell inhibition with imatinib ameliorated cytokine release from skin biopsies and led to a correlative decrease in granulocyte-macrophage colony-stimulating factor and white blood cells in transgenic sickle mice. Targeting mast cells by genetic mutation or pharmacologic inhibition with imatinib ameliorates tonic hyperalgesia and prevents hypoxia/reoxygenation-induced hyperalgesia in sickle mice. Pretreatment with the mast cell stabilizer cromolyn sodium improved analgesia following low doses of morphine that were otherwise ineffective. Mast cell activation therefore underlies sickle pathophysiology leading to inflammation, vascular dysfunction, pain, and requirement for high doses of morphine. Pharmacological targeting of mast cells with imatinib may be a suitable approach to address pain and perhaps treat SCA.

Vanilloid receptor-1 regulates neurogenic inflammation in colon and protects mice from colon cancer.

Neuroinflammation driven by the vanilloid-type ion channel receptor transient receptor potential vanilloid type 1 (TRPV-1) is suspected to play a role in the pathophysiology of inflammatory bowel disease. Because inflammatory bowel disease is known to elevate the risk of colon cancer, we examined postulated roles for TRPV-1-driven neuroinflammation in promoting colitis-associated and spontaneous colon cancer development. Using a well-established model of colitis-associated cancer (CAC), we found that mice genetically deficient in TRPV-1 showed a higher incidence and number of tumors in the distal colon. In like manner, genetic deficiency of TRPV-1 in the APC(Min/+) model of spontaneous colon cancer accentuated the number of colonic adenomas formed. Mechanistic analyses in the CAC model revealed an increased infiltration of inflammatory cells into the tumors along with elevated expression of interleukin (IL)-6 and IL-11 and activation of the STAT3 and NF-κB signaling pathways. Notably, TPRV-1-deficient mice exhibited a defect in expression of the anti-inflammatory neuropeptides, vasoactive intestinal peptide (VIP), and pituitary adenylate cyclase-activating peptide (PACAP) which contributed to the generation of a local proinflammatory environment. Together, our findings argue that by limiting neuroinflammatory processes, TRPV-1 exerts a protective role that restricts the initiation and progression of colon cancer.

Proliferation of sensory C-fibers and subsequent neurogenic inflammation in rat airway induced by inhaled lipopolysaccharide.

Lipopolysaccharide (LPS) is associated with the development and exacerbation of airway inflammation. Increases in innervation of sensory C-fibers and tachykinin receptors, which mainly involve overproduction of neurotrophins such as nerve growth factor (NGF), may enhance neurogenic inflammation. Expression of NGF and its receptors in rat lungs is known to decline with age. We examined whether inhaled LPS causes proliferation of sensory C-fibers, increased expression of tachykinin receptors and subsequent enhancement of neurogenic inflammation in the airways of preweaning rats. Wistar male rats aged 2 weeks inhaled aerosolized LPS derived from Escherichia coli (0.1mg/ml) for 30 min. Evans blue dye leakage into the trachea induced by gaseous formaldehyde or intravenous capsaicin was measured as an index of tachykinin NK1 receptor-mediated vascular permeability. Expression of substance P-immunoreactive nerves, tachykinin NK1 receptors, tumor necrosis factor (TNF)-α and NGF in the trachea was also assessed immunohistochemically. Neurogenic plasma leakage in the trachea increased significantly between 7 and 21 days after LPS inhalation. Expression of TNF-α, NGF, substance P-immunoreactive nerves and tachykinin NK1 receptors was enhanced, peaking at 28 h, 7 days, 14 days and 14 days after LPS inhalation, respectively. Pretreatment with infliximab, a blocking antibody for TNF-α, almost completely abolished the airway changes seen after LPS inhalation. In conclusion, inhaled LPS increased innervation of sensory C-fibers and expression of tachykinin NK1 receptors in the airway, probably resulting in enhancement of neurogenic airway inflammation. These airway responses may be caused by overproduction of neurotrophins including NGF, mainly through a TNF-α-mediated pathway.

Intravesical botulinum toxin A injections plus hydrodistension can reduce nerve growth factor production and control bladder pain in interstitial cystitis.

OBJECTIVES: To investigate the level of nerve growth factor (NGF) mRNA in bladder tissue and the effect of botulinum toxin A (BTX-A) treatment in patients with interstitial cystitis (IC). METHODS: A total of 19 patients with IC were treated with 100 U or 200 U of intravesical BTX-A injections followed by cystoscopic hydrodistension 2 weeks later. Bladder mucosa biopsies were performed before BTX-A injection and immediately after hydrodilation and in 12 controls. The NGF mRNA and protein levels in bladder tissues were assessed by real-time polymerase chain reaction and immunohistochemistry studies to determine differences in NGF expression between patients with IC before and after BTX-A treatment and compare with controls. RESULTS: At 3 months, 14 patients had symptomatic improvement (responders) and 5 did not (nonresponders). The NGF mRNA levels at baseline in the overall IC patient group were significantly greater than those in the controls (0.65 +/- 0.33 versus 0.42 +/- 0.25, P = 0.046). At 2 weeks after BTX-A treatment, the NGF mRNA levels had decreased to 0.47 +/- 0.23 (P = 0.002, compared with baseline) and were no longer significantly different from those of the controls. The NGF mRNA levels decreased significantly in responders and were significantly decreased after BTX-A in 11 patients with a visual analog pain scale reduction of 2 or more. The immunoreactivity study of bladder tissue from patients with IC showed greater NGF density at baseline compared with controls, but the difference was no longer significant after successful BTX-A treatment. CONCLUSIONS: Intravesical BTX-A injections plus hydrodistension reduce bladder pain in patients with IC. The NGF levels in the bladder tissue were significantly increased in patients with IC and decreased to normal level after treatment in responders.

Cyclo-oxygenase and lipoxygenase pathways in mast cell dependent-neurogenic inflammation induced by electrical stimulation of the rat saphenous nerve.

1. We investigated the role of arachidonic acid metabolism and assessed the participation of mast cells and leukocytes in neurogenic inflammation in rat paw skin. We compared the effect of lipoxygenase (LOX) and cyclo-oxygenase (COX) inhibitors on oedema induced by saphenous nerve stimulation, substance P (SP), and compound 48/80. 2. Intravenous (i.v.) pre-treatment with a dual COX/LOX inhibitor (RWJ 63556), a dual LOX inhibitor/cysteinyl-leukotriene (CysLt) receptor antagonist (Rev 5901), a LOX inhibitor (AA 861), a five-lipoxygenase activating factor (FLAP) inhibitor (MK 886), or a glutathione S-transferase inhibitor (ethacrynic acid) significantly inhibited (40 to 60%) the development of neurogenic oedema, but did not affect cutaneous blood flow. Intradermal (i.d.) injection of LOX inhibitors reduced SP-induced oedema (up to 50% for RWJ 63556 and MK 886), whereas ethacrynic acid had a potentiating effect. 3. Indomethacin and rofecoxib, a highly selective COX-2 inhibitor, did not affect neurogenic and SP-induced oedema. Surprisingly, the structurally related COX-2 inhibitors, NS 398 and nimesulide, significantly reduced both neurogenic and SP-induced oedema (70% and 42% for neurogenic oedema, respectively; 49% and 46% for SP-induced oedema, respectively). 4. COX-2 mRNA was undetectable in saphenous nerves and paw skin biopsy samples, before and after saphenous nerve stimulation. 5. A mast cell stabilizer, cromolyn, and a H(1) receptor antagonist, mepyramine, significantly inhibited neurogenic (51% and 43%, respectively) and SP-induced oedema (67% and 63%, respectively). 6. The co-injection of LOX inhibitors and compound 48/80 did not alter the effects of compound 48/80. Conversely, ethacrynic acid had a significant potentiating effect. The pharmacological profile of the effect of COX inhibitors on compound 48/80-induced oedema was similar to that of neurogenic and SP-induced oedema. 7. The polysaccharide, fucoidan (an inhibitor of leukocyte rolling) did not affect neurogenic or SP-induced oedema. 8. Thus, (i) SP-induced leukotriene synthesis is involved in the development of neurogenic oedema in rat paw skin; (ii) this leukotriene-mediated plasma extravasation might be independent of mast cell activation and/or of the adhesion of leukocytes to the endothelium; (iii) COX did not appear to play a significant role in this process.