Injection Of Tumor Necrosis Factor-alpha Research Articles

Microfibril-Associated Protein-3-Like Regulates TGFβ-Induced EMT Process via TNFR2/p38 MAPK Pathway in Endothelial Cells

Abdominal aortic inflammation (AAI) is a major arterial vasculitis characterized by chronic inflammation and fibrosis. Endothelial cells transform into mesenchymal cells (EMT) is one of the significant mechanisms of vasculitis fibrosis. Despite its importance, the molecular mechanism of EMT in AAI remains poorly understood. In this study, we induced AAI in mice through intraperitoneal injection of tumor necrosis factor-alpha (TNFα). To analyze protein expression, we performed Western blotting. Additionally, we extracted RNA using the nanomagnetic bead method to investigate the expression of functionally related genes. We conducted cell migration and invasion assays using scratch and Transwell techniques. Western blot analysis revealed the upregulation of microfibril-associated protein-3-like (MFAP3L) and tumor necrosis factor (TNF) receptor 2 (TNFR2), along with p38 signaling pathway activation. Notably, MFAP3L expression played a crucial role in the transforming growth factor-beta (TGFβ)-induced EMT process in endothelial cells. Furthermore, we identified that MFAP3L-mediated EMT relied on both TNFR2 expression and the activity of the TNFR2/p38 signaling pathway.

MicroRNA‐195 regulates blood pressure by inhibiting NKCC2A

We previously showed that inhibition of renal tumor necrosis factor‐alpha (TNF) production induced by high salt intake increases Na+‐K+‐2Cl−cotransporter isoform A (NKCC2A) mRNA, protein, and phospho (p)NKCC2 expression. MicroRNA (miRNA) contributes to blood pressure regulation, however no studies have addressed the regulation of NKCC2 by miRNA. miR‐195 is one of the most abundant miRNAs expressed in the kidney and qRT‐PCR experiments showed that injection of TNF (5ng/g bw) directly into the kidney of male mice up‐regulates miR‐195 in the renal outer medulla (OM) by approximately 3.5‐fold (p<0.05). Subsequently, a potential miR‐195 binding site in the 3′‐UTR of NKCC2A was identified using the prediction tool TargetScan v5.1, and luciferase reporter gene assays confirmed that NKCC2A mRNA is directly targeted and repressed by miR‐195. Primary cultures of medullary thick ascending limb (mTAL) cells and freshly isolated mTAL tubules express NKCC2 isoforms A and F. However, only miR‐195 and NKCC2A, but not NKCC2F mRNA, were up‐regulated in mTAL cells exposed for 2 h to a change in osmolality from 300 to 500 mOsmol/kgH₂O, produced with NaCl. Transient transfection of mTAL cells with an shRNA vector targeting TNF prevented increases in miR‐195 induced by high NaCl concentration. Moreover, transfection of a miR‐195 mimic into mTAL cells suppressed NKCC2A mRNA, but not NKCC2F mRNA, by approximately 83% (p<0.05) in cells exposed to high NaCl. In contrast, transfection with anti‐miR‐195 increased NKCC2A mRNA without altering NKCC2F mRNA abundance. Laser‐scanning cytometry detected a 1.5 fold (p<0.05) increase in pNKCC2 protein expression in mTAL cells transfected with anti‐miR‐195, and a miR‐195 mimic prevented the increase of pNKCC2 induced by silencing TNF in cells exposed to high NaCl. Next, we determined whether miRNA‐195 regulates HS‐dependent changes in blood pressure by inhibiting NKCC2A. Both TNF and miR‐195 expression increased in OM from mice given 1% NaCl in the drinking water (HS) for 7 days. Intrarenal injection of a lentivirus construct that specifically silenced TNF in the kidney (U6‐TNF‐ex4) inhibited the expression of miR‐195 (p<0.05), while increasing NKCC2A mRNA expression in mice ingesting HS. However, intrarenal injection of murine recombinant TNF significantly upregulated the expression of miR‐195 and suppressed the increases of NKCC2A mRNA in mice ingesting HS. Intrarenal administration of miR‐195 prevented the increase of NKCC2A mRNA abundance in the OM from mice injected with the TNF silencing lentivirus (U6‐TNF‐ex4) compared with the control lentivirus. Moreover, the HS‐induced increase in blood pressure detected by radiotelemetry after renal silencing of TNF was markedly attenuated in mice given an intrarenal injection of miR‐195. Collectively, the study identifies miR‐195 as a salt‐sensitive and TNF inducible miRNA that inhibits HS‐mediated increases in blood pressure by inhibiting NKCC2A.

Nanocarriers Targeting Intercellular Adhesion Molecule Enhance Delivery to the Inflamed Brain

Over 600,000 Americans suffer from ischemic stroke annually. Inflammatory consequences of ischemic stroke include upregulation of adhesion molecules (e.g. intercellular adhesion molecule -1, ICAM) by activated endothelial cells. However, drug delivery to the central nervous system remains an emerging challenge. Here we developed anti-ICAM monoclonal antibody conjugated nanocarriers (ICAM/NCs) to target activated endothelial cells in the pathologically-altered neurovasculature. In a mouse model similar to post-stroke neuroinflammation (intra-striatal injection of Tumor Necrosis Factor-alpha, TNF), ICAM/NCs injected intravenously 2 hrs-post TNF injury accumulated in lungs within 30 min (146.7±2.5 % injected dose per gram tissue (%ID/g)). Over 24 hrs, ICAM/NCs slowly accumulated in the brain, resulting in a >4-fold increase in brain uptake (0.22±0.02%ID/g) vs. 30 minutes (0.05±0.01%ID/g), and significant clearance from lungs (Panel A). In addition, dexamethasone (Dex)-loaded ICAM/NCs were protective against TNF induced brain edema, as measured by extravasation of radiolabeled albumin, while free Dex had no protective effects at the same dose (1.5mg/kg) (Panel B). Flow cytometry of single cell suspensions from lungs and brain in TNF mice revealed that ~80% of ICAM/NC-positive cells were leukocytes in the lungs 30 min-post injection (while in naïve animals, ~40% NC-positive cells were leukocytes), and that ~98% of ICAM/NC-positive cells were leukocytes in the brain 24 hr-post injection (Panel C). The flow cytometry data for the lungs and brain indicated that ICAM/NCs target not only endothelial cells but also leukocytes. Targeting to pulmonary leukocytes is further enhanced following pathological challenges. Altogether, our results suggest that delayed brain delivery of ICAM/NCs was partially due to pulmonary leukocytes ferrying NCs from lungs to brain over time. Future work will focus on elucidating the mechanism of brain delivery.

A Proinflammatory, Degenerative Organ Culture Model to Simulate Early-Stage Intervertebral Disc Disease.

Symptomatic intervertebral disc (IVD) degeneration (IDD) is a major socioeconomic burden and is characterized by inflammation and tissue degradation. Due to the lack of causative therapies, there is an urgent need for innovative experimental organ culture models to study the mechanisms involved in the progression of the disease, find therapeutic targets, and reduce the need for animal models. We here present a novel, three-dimensional organ culture model protocol mimicking the proinflammatory and catabolic microenvironment, which is present during IDD. Initially, bovine caudal IVDs were dissected, cleaned, and cultured in the tissue culture medium. Dynamic physiologic or pathologic loading was applied in a custom-made bioreactor for 2 hours per day. IVDs were assigned to a control group (high glucose medium, physiological loading, phosphate-buffered saline injection) and a pathological group (low glucose medium, pathological loading, tumor necrosis factor-alpha injection) for four days. Gene expression analysis from collected nucleus pulposus cells of the IVDs and enzyme-linked immunosorbent assay of the conditioned organ culture media was performed. Our data revealed a higher expression of inflammatory markers and reduced disc heights after loading in the pathological group compared to the control group. This protocol is reliable to simulate IVD inflammation and degeneration and can be further expanded to broaden its application scope.

YAP1 regulates chondrogenic differentiation of ATDC5 promoted by temporary TNF-α stimulation through AMPK signaling pathway.

Local injection of tumor necrosis factor-alpha (TNF-α) at bone fracture sites during the early stage of the inflammatory response is reported to improve fracture repair in a murine model. However, the underlying mechanism is unclear. Endochondral bone formation, a process that is highly related to fracture repair, requires a certain amount of chondrocyte hypertrophy. This study aimed to investigate the effect of TNF-α on the differentiation of murine chondrogenic ATDC5 cells and the underlying mechanism. In this study, improved chondrogenic differentiation of ATDC5 cells was achieved by brief TNF-α stimulation. Moreover, the expression of Yes-associated protein 1 (YAP1) was suppressed after brief TNF-α stimulation. The expressions of inflammatory mediators and chondrogenic and hypertrophic-associated genes in ATDC5 cells triggered by TNF-α were suppressed in the YAP1 overexpression group but enhanced in the YAP1 knockdown group. Mechanistically, TNF-α-induced activation of the 5' AMP-activated protein kinase (AMPK) signaling pathway was regulated by YAP1, as revealed by the phosphorylated-AMPK/AMPK change ratios in the YAP1 overexpression and knockdown groups, respectively. Moreover, the potential for TNF-α to enhance chondrogenic differentiation could be partially reversed with an AMPK inhibitor. Taken together, we demonstrate, for the first time, that YAP1 modulates the ability of TNF-α to enhance chondrocyte differentiation partly through AMPK signaling.

Renal miR‐195 mediates TNF‐dependent inhibition of NKCC2

We previously showed that tumor necrosis factor‐alpha (TNF) produced by renal epithelial cells inhibits Na+‐K+‐2Cl− cotransporter (NKCC2) activity as part of a mechanism that attenuates increases in blood pressure in response to elevated in sodium intake. However, the mechanisms by which TNF inhibits NKCC2 isoform expression and activity have not been studied. Previous miRNA array studies have found that miR‐195 is one of the most abundant microRNAs expressed in the kidney. In the present study, we demonstrate by qRT‐PCR that TNF up‐regulates mmu‐miR‐146a, ‐195, and ‐203 and down‐regulates mmu‐miR‐30c and ‐100 in the medullary thick ascending limb (mTAL). Most notably, qRT‐PCR analysis indicated that the level of miR‐195 was 4‐fold higher in mTAL tubules isolated from the renal medulla of male mice given a single intrarenal injection of murine recombinant TNF (5ng/g body weight) compared with the control group (saline injection). Data obtained using a TAL‐specific TNF knockout (KO) mouse model showed that miR‐195 abundance in the mTAL tubules from these mice was significantly decreased by 58% (P=0.04, n=4) compared with control mice (floxed TNF littermate controls) in response to ingestion of high salt (HS; 1% NaCl in drinking water) for 3 days. Concomitantly, NKCC2A isoform mRNA abundance was increased 3‐fold while phospho (p)NKCC2 expression increased approximately 64% in the renal medulla of the TAL‐specific TNF KO mice compared with littermate control mice. Collectively, the data suggest that TNF‐derived from the TAL in response to HS intake is an important inducer of miR‐195 levels. Using the prediction tool TargetScan v5.1, we identified a potential miR‐195 binding site in the 3′‐UTR of NKCC2A. Accordingly, intrarenal injection of miR‐195 inhibited the HS‐mediated increases in NKCC2A mRNA and concurrent pNKCC2 expression by 81% and 59%, respectively, in the renal medulla from TAL‐specific TNF KO mice. Consistent with the in vivo effects of TNF on miR‐195, treatment of primary cultures of mouse mTAL cells with 1 nM TNF for 4 hr increased miR‐195 expression more than 3‐fold (P=0.01; n=4). Moreover, NKCC2A mRNA and pNKCC2 expression was inhibited by 76% (P=0.02; n=4) and 61% (P=0.01; n=4), respectively, in mTAL cells exposed to hypertonic conditions (400 mOsmol/kg H2O) following cotreatment with miR‐195 and a short hairpin (sh)TNF silencing lentivirus construct. Silencing of TNFR1 in mTAL cells with a siRNA oligonucleotide inhibited the TNF‐mediated increase of miR‐195 expression and prevented the inhibitory effects of TNF by 73% (P=0.03; n=4), as well as suppressing the increase in pNKCC2 expression. These data identify miR‐195 as an important downstream target of TNFR1‐dependent signaling, which suppresses NKCC2A and pNKCC2 expression in mTAL cells. In summary, the present findings uncover a novel physiological mechanism in which miR‐195 induction via activation of TNFR1‐dependent signaling suppresses NKCC2 expression and activity, suggesting that miR‐195 expression may contribute to the regulation of salt and water excretion and blood pressure via modulation of NKCC2A isoform expression and activity.Support or Funding InformationThis work was supported by NIH grant R01HL133077

Abstract P218: Downregulation of Renal Tumor Necrosis Factor-Alpha Production in Response to Hypotonic Stress Facilitates Adaptive Increases in Renal Cortical Angiotensinogen and Nkcc2b

We previously showed that diverse stimuli activate the thick ascending limb of Henle’s loop (TAL) to synthesize tumor necrosis factor-alpha (TNF), which subsequently acts as an endogenous inhibitor of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC2). Moreover, renal-specific silencing of TNF unmasks salt-dependent increases in blood pressure via a mechanism involving NKCC2A. The objective of the present study was to determine the effects of TNF on NKCC2 isoform and angiotensinogen (AGT) expression in the renal cortex under conditions of hypotonic stress. Mice given a low salt (0.02% NaCl) diet (LS) for 7 days exhibited a 62±7.4% decrease (p<0.5, n=4) in TNF mRNA expression in the renal cortex, compared with mice ingesting a normal salt diet (NSD), which was associated with a concomitant 4-fold increase in renal cortical AGT mRNA accumulation (p<0.05, n=4). Mice ingesting LS also exhibited about a 63% increase (p<0.05, n=4) in cortical TAL phospho-NKCC2 (pNKCC2) expression and a 3-fold increase in NKCC2B mRNA abundance without a concurrent change in NKCC2A mRNA accumulation. The increases in AGT and NKCC2B mRNA abundance were increased by 6-fold (p<0.05, n=4 ) and 5-fold (p<0.05, n=4), respectively, in mice ingesting LS that also received an intrarenal injection of a lentivirus construct designed to specifically silence TNF in the kidney (U6-TNF-ex4) compared with mice injected with control lentivirus (U6). The effects of a single intrarenal injection of murine recombinant TNF (5ng/g body weight) or saline control for 24 hr on renal AGT and NKCC2 mRNA levels were then determined in mice that ingested LS for 7 days. Administration of TNF inhibited the increase of AGT and NKCC2B mRNA abundance by approximately 42±5.9% and 49±6.5% respectively, p<0.05, n=4 ) in mice exposed to hypotonic stress. Similarly, intrarenal injection of TNF inhibited the increase in pNKCC2 by approximately 54% (p<0.05, n=4 ) in renal cortex from mice given LS for 7 days. Collectively, these findings suggest that downregulation of renal TNF production in response to hypotonic conditions contributes to the regulation of sodium chloride reabsorption via adaptive increases in AGT as well as a selective effect on NKCC2B, which is exclusively expressed by macula densa cells in the renal cortex.

Annular puncture with tumor necrosis factor-alpha injection enhances painful behavior with disc degeneration in vivo

Background ContextPainfulintervertebral disc degeneration is extremely common and costly. Effective treatments are lacking because the nature of discogenic pain is complex with limited capacity to distinguish painful conditions from age-related changes in the spine. Hypothesized sources of discogenic pain include chronic inflammation, neurovascular ingrowth, and structural disruption. PurposeThis study aimed to investigate inflammation, pro-neurovascular growth factors, and structural disruption as sources of painful disc degeneration Study Design/SettingThis study used an in vivo study to address these hypothesized mechanisms with anterior intradiscal injections of tumor necrosis factor-alpha (TNFα), pro-neurovascular growth factors: nerve growth factor and vascular endothelial growth factor (NGF and VEGF), and saline with additional sham surgery and naïve controls. Depth of annular puncture was also evaluated for its effects on structural and painful degeneration. MethodsRat lumbar discs were punctured (shallow or deeper puncture) and intradiscally injected with saline, TNFα, or NGF and VEGF. Structural disc degeneration was assessed using X-ray, magnetic resonance imaging (MRI), and histology. The rat painful condition was evaluated using Von Frey hyperalgesia measurements, and substance P immunostaining in dorsal root ganglion (DRG) was performed to determine the source of pain. ResultsSaline injection increased painful responses with degenerative changes in disc height, MRI intensity, and morphologies of disc structure and cell. TNFα and NGF/VEGF accelerated painful behavior, and TNFα-injected animals had increased substance P in DRGs. Deeper punctures led to more severe disc degeneration. Multiple regression analysis showed that the painful behavior was correlated with disc height loss. ConclusionsWe concluded that rate and severity of structural disc degeneration was associated with the amount of annular disruption and puncture depth. The painful behavior was associated with disc height loss and discal inflammatory state, whereas pro-inflammatory cytokines might play a more important role in the level of pain, which might have resulted from enhanced DRG sensitization. These in vivo painful disc degeneration models with different severities of structural changes may be useful for investigating discogenic pain mechanisms and for screening therapies, although interpretations must note the differences between all surgically induced animal models and the human condition.

Effect of Lumbar Intradiscal Injection of Tumor Necrosis Factor-Alpha and Nerve Growth Factor/Vascular Endothelial Growth Factor on Disc Degeneration, Pain Behavior and Neurovascular Ingrowth in an In Vivo Rat Model

Effect of Lumbar Intradiscal Injection of Tumor Necrosis Factor-Alpha and Nerve Growth Factor/Vascular Endothelial Growth Factor on Disc Degeneration, Pain Behavior and Neurovascular Ingrowth in an In Vivo Rat Model

Spinal release of tumour necrosis factor activates c-Jun N-terminal kinase and mediates inflammation-induced hypersensitivity.

BackgroundMounting evidence points to individual contributions of tumour necrosis factor-alpha (TNF) and the c-Jun N-terminal kinase (JNK) pathway to the induction and maintenance of various pain states. Here we explore the role of spinal TNF and JNK in carrageenan-induced hypersensitivity. As links between TNF and JNK have been demonstrated in vitro, we investigated if TNF regulates spinal JNK activity in vivo.MethodsTNF levels in lumbar cerebrospinal fluid (CSF) were measured by enzyme-linked immunosorbent assay, spinal TNF gene expression by real-time polymerase chain reaction and TNF protein expression, JNK and c-Jun phosphorylation by western blotting. The role of spinal TNF and JNK in inflammation-induced mechanical and thermal hypersensitivity was assessed by injecting the TNF inhibitor etanercept and the JNK inhibitors SP600125 and JIP-1 intrathecally (i.t.). TNF-mediated regulation of JNK activity was examined by assessing the effect of i.t. etanercept on inflammation-induced spinal JNK activity.ResultsTNF levels were increased in CSF and spinal cord following carrageenan-induced inflammation. While JNK phosphorylation followed the same temporal pattern as TNF, c-jun was only activated at later time points. Intrathecal injection of TNF and JNK inhibitors attenuated carrageenan-induced mechanical and thermal hypersensitivity. TNF stimulation induced JNK phosphorylation in cultured spinal astrocytes and blocking the spinal actions of TNF in vivo by i.t. injection of etanercept reduced inflammation-induced spinal JNK activity.ConclusionsHere we show that spinal JNK activity is dependent on TNF and that both TNF and the JNK signalling pathways modulate pain-like behaviour induced by peripheral inflammation.

Tonabersat Inhibits Trigeminal Ganglion Neuronal‐Satellite Glial Cell Signaling

Sensitization and activation of trigeminal neurons are implicated in the underlying pathology of migraine, acute sinusitis, and allergic rhinitis. Cell bodies of trigeminal neurons that provide sensory innervation of the dura and nasal mucosa reside in the trigeminal ganglion in association with satellite glial cells where they communicate via gap junctions. Gap junctions, channels formed by connexins, modulate the excitability state of both neurons and glia under pathological conditions. Tonabersat, a compound being tested as an antimigraine drug, is thought to block gap junction activity. To investigate the cellular events within trigeminal ganglia that may account for the significant comorbidity of migraine and rhinosinusitis and determine the effect of tonabersat on neuron-satellite glia communication. Sprague Dawley rats injected with True Blue were used to localize neuronal cell bodies in the ganglion and study neuron-glia signaling via gap junctions in the trigeminal ganglion. Dye coupling studies were conducted under basal conditions and in response to tumor necrosis factor-alpha injection into the whisker pad and/or capsaicin injection into the eyebrow. Changes in connexin 26 and active p38 levels were determined by immunohistochemistry. In addition, the effect of tonabersat prior to chemical stimulation on gap junction activity and expression of connexins and active p38 was investigated. Injection of tumor necrosis factor-alpha, a cytokine implicated in the pathology of acute sinusitis and allergic rhinitis, into the V2 region was shown to lower the amount of capsaicin required to stimulate neurons located in the V1 region of the ganglion. While injection of tumor necrosis factor-alpha into the whisker pad or capsaicin injection into the eyebrow alone did not cause increased dye movement, the combination of both stimuli greatly increased neuron-satellite glia communication via gap junctions in both V1 and V2 regions. The change in gap junction activity was accompanied by increased expression of connexin 26 and active p38 levels in both neurons and satellite glia in V1 and V2 regions. Pretreatment with tonabersat inhibited gap junction communication between neurons and satellite glia and blocked the increase in connexin 26 and active p38 levels in response to injection of both tumor necrosis factor-alpha (V2) and capsaicin (V1). We propose that increased levels of tumor necrosis factor-alpha, as reported during acute sinusitis and allergic rhinitis, reduces the amount of capsaicin necessary to stimulate V1 neurons that leads to cellular changes in both V1 and V2 regions. The cellular events observed in this study may help to explain, in part, the significant comorbidity reported with migraine and rhinosinusitis. In addition, we have provided evidence to suggest that tonabersat can prevent increased neuron-satellite glia signaling and, thus, may be useful in the treatment of migraine, acute sinusitis, and allergic rhinitis.

Antihyperalgesic efficacy of lacosamide in a rat model for muscle pain induced by TNF

Chronic muscle pain is a problem with high prevalence in clinical practice and its pharmacological treatment is difficult. There is a lack of animal models which reliably predict analgesic activity of drugs on muscle pain. Here we used intramuscular injection of tumor necrosis factor-alpha (TNF) in rats as a model of muscle pain. In this model we tested the antihyperalgesic action of lacosamide in comparison to the analgesics pregabalin and gabapentin. Mechanical withdrawal thresholds to muscle pressure were measured with an algesimeter exerting pressure on the gastrocnemius muscles previously injected with TNF. Fore limb grip strength was measured with a digital grip force meter after TNF injection into the biceps brachii muscles. A complete reversal of hyperalgesia was seen with lacosamide at 30mg/kg. Significant effects were also seen for pregabalin at 30 and 100mg/kg and gabapentin at 100mg/kg. In biceps muscle hyperalgesia, a significant reversal of hyperalgesia was seen with lacosamide at 10mg/kg. Significant effects were also seen for pregabalin and gabapentin at 100mg/kg. We could thus demonstrate in a rat model for myalgia that lacosamide effectively reduces muscular hyperalgesia and is somewhat more potent than gabapentin and pregabalin.

Protein kinase C-ζ mediates retinal degeneration in response to TNF

Tumor necrosis factor-alpha (TNF) has been implicated in retinal ganglion cells (RGC) degeneration in glaucoma. Atypical protein kinase C (PKC) ζ is involved in cell protection against various stresses. The aim of this study was to investigate the potential proapoptotic effects of intravitreal injections of TNF with or without PKCζ specific inhibitor on the rat retina. TNF was injected in the vitreous of rat eyes alone or in combination with specific PKCζ inhibitor. PKCζ and NF-κB were studied by immunohistochemistry and western-blotting analysis on retina, and apoptosis quantified by the TUNEL assay. While low basal PKCζ was observed in the control eyes, TNF induced intense expression of PKCζ mostly in bipolar cells processes. PKCζ staining became nuclear when TNF was coinjected with PKCζ inhibitor. TNF alone did not induce apoptosis in the retina. Coinjection of the PKCζ-specific inhibitor and TNF, however, induced apoptosis in the inner nuclear and ganglion cell layers. The PKCζ-specific inhibitor unmasks retinal cells to TNF cytotoxicity showing a link between the proapoptotic effects of TNF and the antiapoptotic PKCζ signaling pathway.

Participation of the Endocannabinoid System in the Effect of TNF‐α on Hypothalamic Release of Gonadotropin‐Releasing Hormone

It is known that Delta(9)-tetrahydrocannabinol (THC), the major active ingredient of marijuana, can suppress reproductive function. Also, we reported previously that the endocannabinoid, anandamide (AEA), inhibited gonadotropin-releasing hormone (LHRH) release from medial basal hypothalamus (MBH) of male rats incubated in vitro as well as reduced plasma LH levels after i.c.v. AEA injections into the cerebral lateral ventricle. On the other hand, it is known that during endotoxemia the hypothalamic gonadotropin axis is inhibited. Therefore, the aim of the present study was to determine whether the effect of TNF-alpha, a proinflammatory cytokine induced by lipopolysaccharide (LPS) that inhibits LHRH release, is mediated by the activation of the endocannabinoid system. The intraperitoneal injection of LPS (5 mg/kg) as well as the i.c.v. injection of tumor necrosis factor-alpha (TNF-alpha) (100 ng/rat) increased significantly the AEA synthesis measured ex vivo in MBHs removed 3 h after the treatments. To examine the possibility that TNF-alpha also acted by increasing the synthesis of AEA that was released and activated the CB1-r followed by inhibition of LHRH release, we measured the effect of TNF-alpha on the AEA synthase activity in MBHs incubated in vitro. As expected, we found that TNF-alpha (2.9 x 10(-9) M) increased the AEA synthesis. Second, we showed that TNF-alpha reduced significantly the forskolin-stimulated LHRH release and that the CB1-r antagonist AM251 (10(-5) M) blocked that inhibition, supporting the hypothesis that TNF-alpha inhibits LHRH release, acting at least in part by activating the endocannabinoid system. Therefore, our data demonstrate a key role for the endocannabinoid system in the response of the reproductive system to inflammatory signals.

(625): Lacosamide displays antinociceptive effects in a rat model for musculoskelatal pain induced by TNF

Chronic musculoskeletal pain which is not traced to a specific structural or inflammatory cause occurs with fibromyalgia, myofacial pain syndromes or back pain. The goal in treating musculoskeletal pain is to decrease pain and to increase muscular function. Non steroidal anti-inflammatory drugs, muscle relaxants, and benzodiazepines are all frequently used musculoskeletal pain treatments, but none are particularly helpful in chronic conditions clearly indicating a need for improved treatment modalities. Here we used intramuscular injection of tumor necrosis factor-alpha (TNF) in rats as a model of muscle pain. In this model we tested the antinociceptive action of the new anticonvulsant lacosamide (3, 10, 30 mg/kg) in comparison to the anticonvulsant gabapentin (100 mg/kg) and to metamizol (2 mg/kg). Mechanical withdrawal thresholds to muscle pressure were measured with an algesimeter exerting pressure on the gastrocnemius muscle previously injected with TNF. Forelimb grip strength was measured with a digital grip force meter after TNF injection into the biceps brachii muscles. A complete reversal of hyperalgesia was seen with lacosamide at 30 mg/kg. Significant effects were also seen for gabapentin and metamizol. In biceps muscle hyperalgesia, a significant inhibition of hyperalgesia was seen with lacosamide at 10 mg/kg as for gabapentin and metamizol. We could thus demonstrate in a rat model for myalgia that lacosamide effectively reduces muscular hyperalgesia and is somewhat more efficaceous than gabapentin and metamizol. Chronic musculoskeletal pain which is not traced to a specific structural or inflammatory cause occurs with fibromyalgia, myofacial pain syndromes or back pain. The goal in treating musculoskeletal pain is to decrease pain and to increase muscular function. Non steroidal anti-inflammatory drugs, muscle relaxants, and benzodiazepines are all frequently used musculoskeletal pain treatments, but none are particularly helpful in chronic conditions clearly indicating a need for improved treatment modalities. Here we used intramuscular injection of tumor necrosis factor-alpha (TNF) in rats as a model of muscle pain. In this model we tested the antinociceptive action of the new anticonvulsant lacosamide (3, 10, 30 mg/kg) in comparison to the anticonvulsant gabapentin (100 mg/kg) and to metamizol (2 mg/kg). Mechanical withdrawal thresholds to muscle pressure were measured with an algesimeter exerting pressure on the gastrocnemius muscle previously injected with TNF. Forelimb grip strength was measured with a digital grip force meter after TNF injection into the biceps brachii muscles. A complete reversal of hyperalgesia was seen with lacosamide at 30 mg/kg. Significant effects were also seen for gabapentin and metamizol. In biceps muscle hyperalgesia, a significant inhibition of hyperalgesia was seen with lacosamide at 10 mg/kg as for gabapentin and metamizol. We could thus demonstrate in a rat model for myalgia that lacosamide effectively reduces muscular hyperalgesia and is somewhat more efficaceous than gabapentin and metamizol.

Differential Modulatory Effects of Cholera Toxin and Pertussis Toxin on Pain Behavior Induced by TNF-a, lnterleukin-1β and Interferon- Injected Intrathecally

The present study was designed to characterize the possible roles of spinally located cholera toxin (CTX)- and pertussis toxin (PTX)-sensitive G-proteins in pro-inflammatory cytokine induced pain behaviors. Intrathecal injection of tumor necrosis factor-alpha (TNF-alpha; 100 pg), interleukin-1beta (IL-1beta; 100 pg) and interferon-gamma (INF-gamma; 100 pg) showed pain behavior. Intrathecal pretreatment with CTX (0.05, 0.1 and 0.5 mg) attenuated pain behavior induced by TNF-alpha and INF-gamma administered intrathecally. But intrathecal pretreatment with CTX (0.05, 0.1 and 0.5 microg) did not attenuate pain behavior induced by IL-1beta. On the other hand, intrathecal pretreatment with PTX further increased the pain behavior induced by TNF-alpha and IL-1beta administered intrathecally, especially at the dose of 0.5 microg. But intrathecal pretreatment with PTX did not affect pain behavior induced by INF-gamma. Our results suggest that, at the spinal cord level, CTX- and PTX-sensitive G-proteins appear to play important roles in modulating pain behavior induced by pro-inflammatory cytokines administered spinally. Furthermore, TNF-alpha, IL-1beta and INF-gamma administered spinally appear to produce pain behavior by different mechanisms.

Dual role of tumor necrosis factor-alpha in hepatic ischemia-reperfusion injury: studies in tumor necrosis factor-alpha gene knockout mice.

Although hepatic ischemia-reperfusion (IR) injury is partially mediated by tumor necrosis factor-alpha (TNF), we recently found that low-dose TNF before IR is hepatoprotective. We examined the seemingly conflicting roles of TNF in mediating liver injury in a partial hepatic IR model using TNF gene knockout (TNF ko) mice to allow TNF replacement at specified times. Compared with wild-type mice, TNF ko mice exhibit minimal alanine aminotransferase release and few hepatonecrotic lesions during the early (time, 2 hours) and late (time, 24 hours) phases of IR. TNF ko mice differed from wild-type mice in that TNF ko mice exhibited no activation or induction of nuclear factor-kappa B, p38, cyclin D1, or proliferating cell nuclear antigen after IR. A single low-dose TNF injection 1 minute before the onset of hepatic ischemia restored hepatic IR injury in TNF ko mice. To clarify the importance of TNF for hepatoprotection, preconditioning (10 minutes of ischemia and 10 minutes of reperfusion) was performed before the onset of IR for TNF ko mice whose capacity to undergo IR injury had been restored by TNF replacement. Ischemic preconditioning failed to protect these mice from TNF-augmented IR injury; however, following the administration of intravenous TNF (1 microg per kg body weight, which mimics the early increase in hepatic and plasma TNF levels that is mobilized by ischemic preconditioning), significant hepatoprotection against both the early and late phases of TNF-augmented IR injury was observed. In conclusion, TNF appears to mediate both the early and late phases of liver injury in hepatic IR, but it also is an essential mediator of hepatoprotective effects brought about by ischemic preconditioning.

Cyclooxygenase inhibition in nerve-injury- and TNF-induced hyperalgesia in the rat

After nerve injury, cyclooxygenase-2 (COX-2) is upregulated in spinal cord and peripheral nerve, the latter being dependent on tumor necrosis factor-alpha (TNF). Here we asked whether COX inhibitors attenuate pain behavior induced by chronic constrictive sciatic nerve injury (CCI) or intraneural injection of TNF (2.5 pg/ml). Rats received either 0.9% saline, the nonselective COX inhibitor ibuprofen (40 mg/kg) or the selective COX-2 inhibitor celecoxib (10 or 30 mg/kg) twice daily by gavage started 2 days before, 12 h or 7 days after surgery. Mechanical allodynia and thermal hyperalgesia induced by CCI was moderately, but consistently attenuated by early (day −2 or 12 h after CCI), but not late (7 days after CCI) ibuprofen and celecoxib treatment. Mechanical allodynia, but not thermal hyperalgesia induced by intraneural TNF, was reduced by ibuprofen, but not by celecoxib treatment 5 and 7 days after injection. Sciatic nerves, lumbar dorsal root ganglia (DRG) and spinal cords from rats with treatment started 12 h after surgery were analyzed for prostaglandin E 2 (PGE 2) levels 10 days after CCI. In injured nerves and ipsilateral DRG, PGE 2 levels were increased. Ibuprofen treatment reversed PGE 2 levels in injured nerves and DRG, whereas celecoxib blocked increased PGE 2 levels only in nerves. In spinal cord, no change in PGE 2 levels was observed. In contrast to the marked inhibition of nerve-injury-induced upregulation of PGE 2 by COX inhibitors, the effect on pain behavior was modest. Nerve-injury- and TNF-induced pain-related behavior seem to be only partly dependent on peripheral prostaglandins.

TNF-alpha microinjection upregulates chemokines and chemokine receptors in the central nervous system without inducing leukocyte infiltration.

Chemokines (chemoattractant cytokines) are key players in the initiation of inflammatory cell accumulation in the central nervous system (CNS). Mechanisms leading to upregulation of chemokines in CNS pathologic conditions remain largely unknown. Numerous in vitro studies showed that inflammatory cytokines stimulate cultured CNS cells to produce chemokines. The main goal of this study was to analyze if an individual proinflammatory cytokine is sufficient to upregulate the chemokine system in the adult CNS in vivo. We analyzed CC chemokine ligand and receptor expression in brains from two different strains of mice (SJL and BALB) after stereotaxic, intracerebral injection of tumor necrosis factor-alpha (TNF-alpha). In both strains, we detected similarly increased expression of chemokines RANTES/CCL5, macrophage inflammatory protein-1alpha (MIP-1alpha)/CCL3, MIP-1beta/CCL4, and MIP-2, as well as chemokine receptors CCR1, CCR2, and CCR5. Interestingly, we did not observe parenchymal leukocyte infiltrates after local TNF-alpha delivery. This observation shows that upregulation of chemokines by TNF-alpha is not sufficient to cause accumulation of leukocytes in the CNS parenchyma in both strains of mice.

Intramuscular injection of tumor necrosis factor-alpha induces muscle hyperalgesia in rats

The role of proinflammatory cytokines in neuropathic and inflammatory pain is well established. Recent studies suggest that cytokines such as tumor necrosis factor-alpha (TNF) may also be involved in the development of muscle pain. To investigate the pathophysiology of intramuscular TNF, exogenous TNF (0.1–10 μg), formalin (9%) or vehicle was injected into the gastrocnemius or biceps brachii muscles of rats. To quantify muscle hyperalgesia, changes in forelimb grip force or withdrawal thresholds to increasing pressure applied to the gastrocnemius muscle were measured. TNF evoked a time- and dose-dependent muscle hyperalgesia within several hours after injection that was totally reversed by systemic treatment with the non-opioid analgesic metamizol. Paw withdrawal thresholds or latencies to mechanical and thermal stimuli, respectively, were unchanged after intramuscular injection of TNF or formalin. In contrast to formalin, which induced significant muscle tissue damage, macrophage infiltration, swelling and partial motor impairment demonstrated in rotarod tests, TNF induced neither histopathological tissue damage nor motor dysfunction. To investigate the effect of TNF and formalin on other potentially algesic mediators, muscles were analyzed for calcitonin-gene related peptide (CGRP), prostaglandin E 2 (PGE 2) and nerve growth factor (NGF) 1 day after injection. TNF and formalin evoked intramuscular upregulation of CGRP and NGF, whereas PGE 2 was increased exclusively after TNF injection. These findings allow us to speculate that endogenous TNF may play a role in the development of muscle hyperalgesia. Targeting proinflammatory cytokines might be beneficial for the treatment of musculoskeletal pain syndromes.