Proteomics reveals that a high-fat diet induces rapid changes in hypothalamic proteins related to neuronal damage and inflammation

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The p75NTR SIGNALING CASCADE MEDIATES MECHANICAL HYPERALGESIA INDUCED BY NERVE GROWTH FACTOR INJECTED INTO THE RAT HIND PAW

Nerve Growth Factor (NGF) augments excitability of isolated rat sensory neurons through activation of the p75 neurotrophin receptor (p75NTR) and its downstream sphingomyelin signaling cascade, wherein neutral sphingomyelinase(s) (nSMase), ceramide, and the atypical PKC (aPKC), PKMζ, are key mediators. Here we examined these same receptor-pathways in vivo for their role in mechanical hyperalgesia from exogenous NGF. Mechanical sensitivity was tested by the number of paw withdrawals in response to 10 stimuli (PWF = n/10) by a 4g von Frey hair (VFH, testing “allodynia”) and by 10g and 15g VFHs (testing “hyperalgesia”). NGF (500 ng/10 µl) injected into the male rat’s plantar hind paw induced long lasting ipsilateral mechanical hypersensitivity. Mechano-hypersensitivity, relative to baseline responses and to those of the contralateral paw, developed by 0.5–1.5h and remained elevated at least for 21–24h, Acute intraplantar pre-treatment with nSMase inhibitors, GSH or GW4869, prevented the acute hyperalgesia from NGF (at 1.5h) but not that at 24h. A single injection of N-acetyl sphingosine (C2-ceramide), simulating the ceramide produced by nSMase activity, induced ipsilateral allodynia that persisted for 24h, and transient hyperalgesia that resolved by 2h. Intraplantar injection of hydrolysis-resistant mPro-NGF, selective for the p75NTR over the TrkA receptor, gave very similar results to NGF and was susceptible to the same inhibitors. Hyperalgesia from both NGF and mPro-NGF was prevented by paw pre-injection with blocking antibodies to rat p75NTR receptor. Finally, intraplantar (1 day before NGF) injection of mPSI, the myristolated pseudosubstrate inhibitor of PKCζ/PKMζ, decreased the hyperalgesia resulting from NGF or C2-ceramide, although scrambled mPSI was ineffective. The findings indicate that mechano-hypersensitivity from peripheral NGF involves the sphingomyelin signaling cascade activated via p75NTR, and that a peripheral aPKC is essential for this sensitization

The TrkA receptor mediates experimental thermal hyperalgesia produced by nerve growth factor: Modulation by the p75 neurotrophin receptor

The p75 neurotrophin receptor (p75NTR) and its activation of the sphingomyelin signaling cascade are essential for mechanical hypersensitivity resulting from locally injected nerve growth factor (NGF). Here the roles of the same effectors, and of the tropomyosin receptor kinase A (TrkA) receptor, are evaluated for thermal hyperalgesia from NGF. Sensitivity of rat hind paw plantar skin to thermal stimulation after local sub-cutaneous injection of NGF (500ng) was measured by the latency for paw withdrawal (PWL) from a radiant heat source. PWL was reduced from baseline values at 0.5-22h by ∼40% from that in naïve or vehicle-injected rats, and recovered to pre-injection levels by 48h. Local pre-injection with a p75NTR blocking antibody did not affect the acute thermal hyperalgesia (0.5-3.5h) but hastened its recovery so that it had reversed to baseline by 22h. In addition, GW4869 (2mM), an inhibitor of the neutral sphingomyelinase (nSMase) that is an enzyme in the p75NTR pathway, also failed to prevent thermal hyperalgesia. However, C2-ceramide, an analog of the ceramide produced by sphingomyelinase, did cause thermal hyperalgesia. Injection of an anti-TrkA antibody known to promote dimerization and activation of that receptor, independent of NGF, also caused thermal hyperalgesia, and prevented the further reduction of PWL from subsequently injected NGF. A non-specific inhibitor of tropomyosin receptor kinases, K252a, prevented thermal hyperalgesia from NGF, but not that from the anti-TrkA antibody. These findings suggest that the TrkA receptor has a predominant role in thermal hypersensitivity induced by NGF, while p75NTR and its pathway intermediates serve a modulatory role

Nerve growth factor/p75 neurotrophin receptor–mediated sensitization of rat sensory neurons depends on membrane cholesterol

Nerve growth factor (NGF) is an important mediator in the initiation of the inflammatory response and NGF via activation of the p75 neurotrophin receptor (p75(NTR)) and downstream sphingomyelin signaling leads to significant enhancement of the excitability of small-diameter sensory neurons. Because of the interaction between sphingomyelin and cholesterol in creating membrane liquid-ordered domains known as membrane or lipid rafts, we examined whether neuronal NGF-induced sensitization via p75(NTR) was dependent on the integrity of membrane rafts. Here, we demonstrate that the capacity of NGF to enhance the excitability of sensory neurons may result from the interaction of p75(NTR) with its downstream signaling partner(s) in membrane rafts. Two agents known to disrupt membrane rafts, edelfosine and methyl-β-cyclodextrin (MβCD), block the increase in excitability produced by NGF. In contrast, treatment with MβCD containing saturated amounts of cholesterol does not alter the capacity of NGF to augment excitability. In addition, adding back MβCD with cholesterol restored the NGF-induced sensitization in previously cholesterol-depleted neurons, suggesting that cholesterol and the structural integrity of rafts are key to promoting NGF-mediated sensitization. Using established protocols to isolate detergent-resistant membranes, both p75(NTR) and the neuronal membrane raft marker, flotillin, localize to raft fractions. These results suggest that downstream signaling partners interacting with p75(NTR) in sensory neurons are associated with membrane raft signaling platforms

Tumor necrosis factor enhances the capsaicin sensitivity of rat sensory neurons

The capacity of the proinflammatory cytokines, tumor necrosis factor alpha (TNF alpha) and interleukin 1 beta (IL-1 beta), to modulate the sensitivity of isolated sensory neurons grown in culture to the excitatory chemical agent capsaicin was examined. Alterations in capsaicin sensitivity were assessed by quantifying the number of neurons labeled with cobalt after exposure to capsaicin and by recording the whole-cell response from a single neuron to the focal application of capsaicin. A 24 hr pretreatment of the neuronal cultures with TNF alpha (10 or 50 ng/ml), but not IL-1 beta (10 or 50 ng/ml), produced a concentration-dependent increase in the number of cobalt-labeled neurons after exposure to 100 nM capsaicin. The peak increase in the number of labeled neurons was attained after a 4 hr treatment with 10 ng/ml TNF alpha. Similarly, pretreatment with TNF alpha (10 ng/ml for 4, 12, and 24 hr) produced a greater than twofold increase in the average peak amplitude of the inward current evoked by 100 nM capsaicin. Both the TNF alpha-induced increase in labeling and current amplitude were blocked by treating the neuronal cultures with indomethacin before the addition of TNF alpha. Enhancement of the capsaicin-evoked current also was blocked by the specific cyclo-oxygenase-2 inhibitor SC-236. These results indicate that TNF alpha can enhance the sensitivity of sensory neurons to the excitation produced by capsaicin and that this enhancement likely is mediated by the neuronal production of prostaglandins. Isolated sensory neurons grown in culture may prove to be a useful model system in which to explore how prolonged exposure to mediators associated with chronic inflammation alter the regulatory pathways that modulate the excitability of the nervous system

Liveweight gain per head and per ha throughout the year of lambs grazing conventional pastures and those that switch from grass to clover

Intensive lamb finishing requires a consistent supply of high quality forage throughout the year to regularly finish lambs. Per head and per ha liveweight gain of weaned lambs was compared in 13 batches of lambs on replicated irrigated farmlets for 2.5 years from conventional mixed tetraploid perennial ryegrasswhite clover pastures (Conv) and pastures that were pure white clover for spring and summer and switched to overdrilled Italian ryegrass for the winter (Switch). Seasonal differences in stocking rate (lambs/ha), liveweight gain per head and per ha were significant (P<0.05). Average daily liveweight gain/ha was significantly higher (6.01 versus 5.66 kg/ha/day for Switch and Conv, respectively, but the total grazing days were slightly lower on the Switch farmlets resulting in similar annualised liveweight gain per ha (1 800 kg) and net carcass weight (800 kg/ha) on both pasture treatments. The farmlets apparently utilised 10 000 kg DM/ha/yr of the 16 000 kg DM accumulated.This work was funded by an internal Lincoln University Research Grant INT4052

Sphingosine 1-phosphate enhances the excitability of rat sensory neurons through activation of sphingosine 1-phosphate receptors 1 and/or 3

BACKGROUND: Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that acts through a family of five G-protein-coupled receptors (S1PR1-5) and plays a key role in regulating the inflammatory response. Our previous studies demonstrated that rat sensory neurons express the mRNAs for all five S1PRs and that S1P increases neuronal excitability primarily, but not exclusively, through S1PR1. This raises the question as to which other S1PRs mediate the enhanced excitability. METHODS: Isolated sensory neurons were treated with either short-interfering RNAs (siRNAs) or a variety of pharmacological agents targeted to S1PR1/R2/R3 to determine the role(s) of these receptors in regulating neuronal excitability. The excitability of isolated sensory neurons was assessed by using whole-cell patch-clamp recording to measure the capacity of these cells to fire action potentials (APs). RESULTS: After siRNA treatment, exposure to S1P failed to augment the excitability. Pooled siRNA targeted to S1PR1 and R3 also blocked the enhanced excitability produced by S1P. Consistent with the siRNA results, pretreatment with W146 and CAY10444, selective antagonists for S1PR1 and S1PR3, respectively, prevented the S1P-induced increase in neuronal excitability. Similarly, S1P failed to augment excitability after pretreatment with either VPC 23019, which is a S1PR1 and R3 antagonist, or VPC 44116, the phosphonate analog of VPC 23019. Acute exposure (10 to 15 min) to either of the well-established functional antagonists, FTY720 or CYM-5442, produced a significant increase in the excitability. Moreover, after a 1-h pretreatment with FTY720 (an agonist for S1PR1/R3/R4/R5), neither SEW2871 (S1PR1 selective agonist) nor S1P augmented the excitability. However, after pretreatment with CYM-5442 (selective for S1PR1), SEW2871 was ineffective, but S1P increased the excitability of some, but not all, sensory neurons. CONCLUSIONS: These results demonstrate that the enhanced excitability produced by S1P is mediated by activation of S1PR1 and/or S1PR3

Peripheral Synthesis of an Atypical Protein Kinase C Mediates the Enhancement of Excitability and the Development of Mechanical Hyperalgesia Produced by Nerve Growth Factor

Nerve growth factor (NGF) plays a key role in the initiation as well as the prolonged heightened pain sensitivity of the inflammatory response. Previously, we showed that NGF rapidly augmented both the excitability of isolated rat sensory neurons and the mechanical sensitivity of the rat’s hind paw. The increase in excitability and sensitivity was blocked by the myristoylated pseudosubstrate inhibitor of atypical PKCs (mPSI), suggesting that an atypical PKC may play a key regulatory role in generating this heightened sensitivity. Our findings raised the question as to whether NGF directs changes in translational control, as suggested for long-lasting long-term potentiation (LTP), or whether NGF leads to the activation of an atypical PKC by other mechanisms. The current studies demonstrate that enhanced action potential (AP) firing produced by NGF was blocked by inhibitors of translation, but not transcription. In parallel, in vitro studies showed that NGF elevated the protein levels of PKMζ, which was also prevented by inhibitors of translation. Intraplantar injection of NGF in the rat hind paw produced a rapid and maintained increase in mechanical sensitivity whose onset was delayed by translation inhibitors. Established NGF-induced hypersensitivity could be transiently reversed by injection of rapamycin or mPSI. These results suggest that NGF produces a rapid increase in the synthesis of PKMζ protein in the paw that augments neuronal sensitivity and that the ongoing translational expression of PKMζ plays a critical role in generating as well as maintaining the heightened sensitivity produced by NGF

Delays and loss to follow-up before treatment of drug-resistant tuberculosis following implementation of Xpert MTB/RIF in South Africa: A retrospective cohort study.

BACKGROUND: South Africa has a large burden of rifampicin-resistant tuberculosis (RR-TB), with 18,734 patients diagnosed in 2014. The number of diagnosed patients has increased substantially with the introduction of the Xpert MTB/RIF test, used for tuberculosis (TB) diagnosis for all patients with presumptive TB. Routine aggregate data suggest a large treatment gap (pre-treatment loss to follow-up) between the numbers of patients with laboratory-confirmed RR-TB and those reported to have started second-line treatment. We aimed to assess the impact of Xpert MTB/RIF implementation on the delay to treatment initiation and loss to follow-up before second-line treatment for RR-TB across South Africa. METHODS AND FINDINGS: A nationwide retrospective cohort study was conducted to assess second-line treatment initiation and treatment delay among laboratory-diagnosed RR-TB patients. Cohorts, including approximately 300 sequentially diagnosed RR-TB patients per South African province, were drawn from the years 2011 and 2013, i.e., before and after Xpert implementation. Patients with prior laboratory RR-TB diagnoses within 6 mo and currently treated patients were excluded. Treatment initiation was determined through data linkage with national and local treatment registers, medical record review, interviews with health care staff, and direct contact with patients or household members. Additional laboratory data were used to track cases. National estimates of the percentage of patients who initiated treatment and time to treatment were weighted to account for the sampling design. There were 2,508 and 2,528 eligible patients in the 2011 and 2013 cohorts, respectively; 92% were newly diagnosed with RR-TB (no prior RR-TB diagnoses). Nationally, among the 2,340 and 2,311 new RR-TB patients in the 2011 and 2013 cohorts, 55% (95% CI 53%-57%) and 63% (95% CI 61%-65%), respectively, started treatment within 6 mo of laboratory receipt of their diagnostic specimen (p < 0.001). However, in 2013, there was no difference in the percentage of patients who initiated treatment at 6 mo between the 1,368 new RR-TB patients diagnosed by Xpert (62%, 95% CI 59%-65%) and the 943 diagnosed by other methods (64%, 95% CI 61%-67%) (p = 0.39). The median time to treatment decreased from 44 d (interquartile range [IQR] 20-69) in 2011 to 22 d (IQR 2-43) in 2013 (p < 0.001). In 2013, across the nine provinces, there were substantial variations in both treatment initiation (range 51%-73% by 6 mo) and median time to treatment (range 15-36 d, n = 1,450), and only 53% of the 1,448 new RR-TB patients who received treatment were recorded in the national RR-TB register. This retrospective study is limited by the lack of information to assess reasons for non-initiation of treatment, particularly pre-treatment mortality data. Other limitations include the use of names and dates of birth to locate patient-level data, potentially resulting in missed treatment initiation among some patients. CONCLUSIONS: In 2013, there was a large treatment gap for RR-TB in South Africa that varied significantly across provinces. Xpert implementation, while reducing treatment delay, had not contributed substantially to reducing the treatment gap in 2013. However, given improved case detection with Xpert, a larger proportion of RR-TB patients overall have received treatment, with reduced delays. Nonetheless, strategies to further improve linkage to treatment for all diagnosed RR-TB patients are urgently required

ECG findings in professional rugby players using international screening recommendations.

BackgroundWhile World Rugby guidelines do not mandate the inclusion of an electrocardiogram (ECG) for all players, this is required for entry into international rugby competitions. We, therefore, sought to describe sport-specific normative ECG values and evaluate the performance of contemporary athlete ECG guidelines in male and female professional rugby players.MethodsWe retrospectively analysed professional rugby players' ECGs (n=356, male 79%) obtained during preparticipation screening (2010-2022), comparing by sex and playing position (forwards vs backs). ECGs were categorised as normal 'training-related', borderline and abnormal findings, as defined by the 2017 International Recommendations.Results84% of players had one or more normal, 'training-related' findings, with males having a higher prevalence than females (91% vs 60%, pConclusionsThe application of contemporary ECG interpretation criteria resulted in a low positivity rate isolated to male players. These results help inform the logistic feasibility of ECG-inclusive screening, which is already required to enter major tournaments