PTX-treated Rats Research Articles

Melatonin Relieves Paclitaxel-Induced Neuropathic Pain by Regulating pNEK2-Dependent Epigenetic Pathways in DRG Neurons.

The neurohormone melatonin (MLT) demonstrates promising potential in ameliorating neuropathic pain induced by paclitaxel (PTX) chemotherapy. However, little is known about its protective effect on dorsal root ganglion (DRG) neurons in neuropathic pain resulting from the chemotherapeutic drug PTX. Here, PTX-treated rats revealed that intrathecal administration of MLT dose-dependently elevated hind paw withdrawal thresholds and latency, indicating that MLT significantly reversed PTX-induced neuropathic pain. Mechanistically, the analgesic effects of MLT were found to be mediated via melatonin receptor 2 (MT2), as pretreatment with an MT2 receptor antagonist inhibited these effects. Moreover, intrathecal MLT injection reversed the pNEK2-dependent epigenetic program induced by PTX. All of the effects caused by MLT were blocked by pretreatment with an MT2 receptor-selective antagonist, 4P-PDOT. Remarkably, multiple MLT administered during PTX treatment (PTX+MLTs) exhibited not only rapid but also lasting reversal of allodynia/hyperalgesia compared to single-bolus MLT administered after PTX treatment (PTX+MLT). In addition, PTX+MLTs exhibited greater efficacy in reversing PTX-induced alterations in pRSK2, pNEK2, JMJD3, H3K27me3, and TRPV1 expression and interaction in DRG neurons than PTX+MLT. These results indicated that MLT administered during PTX treatment reduced the incidence and/or severity of neuropathy and had a better inhibitory effect on the pNEK2-dependent epigenetic program compared to MLT administered after PTX treatment. In conclusion, MLT/MT2 is a promising therapy for the treatment of pNEK2-dependent painful neuropathy resulting from PTX treatment. MLT administered during PTX chemotherapy may be more effective in the prevention or reduction of PTX-induced neuropathy and maintaining quality.

Phosphate NIMA-Related Kinase 2-Dependent Epigenetic Pathways in Dorsal Root Ganglion Neurons Mediates Paclitaxel-Induced Neuropathic Pain.

The microtubule-stabilizing drug paclitaxel (PTX) is an important chemotherapeutic agent for cancer treatment and causes peripheral neuropathy as a common side effect that substantially impacts the functional status and quality of life of patients. The mechanistic role for NIMA-related kinase 2 (NEK2) in the progression of PTX-induced neuropathic pain has not been established. Adult male Sprague-Dawley rats intraperitoneally received PTX to induce neuropathic pain. The protein expression levels in the dorsal root ganglion (DRG) of animals were measured by biochemical analyses. Nociceptive behaviors were evaluated by von Frey tests and hot plate tests. PTX increased phosphorylation of the important microtubule dynamics regulator NEK2 in DRG neurons and induced profound neuropathic allodynia. PTX-activated phosphorylated NEK2 (pNEK2) increased jumonji domain-containing 3 (JMJD3) protein, a histone demethylase protein, to specifically catalyze the demethylation of the repressive histone mark H3 lysine 27 trimethylation (H3K27me3) at the Trpv1 gene, thereby enhancing transient receptor potential vanilloid subtype-1 (TRPV1) expression in DRG neurons. Moreover, the pNEK2-dependent PTX response program is regulated by enhancing p90 ribosomal S6 kinase 2 (RSK2) phosphorylation. Conversely, intrathecal injections of kaempferol (a selective RSK2 activation antagonist), NCL 00017509 (a selective NEK2 inhibitor), NEK2-targeted siRNA, GSK-J4 (a selective JMJD3 inhibitor), or capsazepine (an antagonist of TRPV1 receptor) into PTX-treated rats reversed neuropathic allodynia and restored silencing of the Trpv1 gene, suggesting the hierarchy and interaction among phosphorylated RSK2 (pRSK2), pNEK2, JMJD3, H3K27me3, and TRPV1 in the DRG neurons in PTX-induced neuropathic pain. pRSK2/JMJD3/H3K27me3/TRPV1 signaling in the DRG neurons plays as a key regulator for PTX therapeutic approaches.

Resistance of Food-Maintained Operant Responding to Mechanical Punishment in Rats: Further Evidence for Weak "Affective/Motivational Pain" in Rat Models of Inflammatory and Neuropathic Pain.

Clinically relevant chronic pain is often associated with functional impairment and behavioral depression as an “affective/motivational” sign of pain; however preclinical animal models of inflammatory and neuropathic pain often produce weak evidence of impaired function. We hypothesized that hindpaw mechanical stimulation produced by a requirement to rear on a textured “NOX” plate would punish operant responding in rats treated with intraplantar complete Freund’s adjuvant (CFA, a model of inflammatory pain) or the chemotherapeutic paclitaxel (PTX, a model of neuropathic pain) and produce sustained pain-related depression of operant behavior. Male Sprague–Dawley rats were trained under a progressive-ratio (PR) schedule of food-maintained operant responding, then treated with CFA (100 µL in left hindpaw), PTX (2.0 mg/kg IP on alternate days for four total injections; 6.6 mg/kg IV on alternate days for three total injections), or saline vehicle. PR break points and mechanical thresholds for paw withdrawal from von Frey filaments were then tracked for 28 days. Subsequently, rats were tested with the opioid receptor antagonist naltrexone to assess latent sensitization and with the kappa opioid receptor (KOR) agonist U69593 to assess KOR function. CFA produced significant mechanical hypersensitivity for 3 weeks but decreased PR breakpoints for only 1 day. Both IP and IV PTX produced mechanical hypersensitivity for at least three weeks; however, only IV PTX decreased PR breakpoints, and this decrease was not alleviated by morphine. After recovery, naltrexone reinstated mechanical hypersensitivity in CFA- but not PTX-treated rats, and it did not reinstate depression of breakpoints in any group. U69593 dose-dependently decreased PR breakpoints in all groups with no difference between control vs. CFA/PTX groups. These results suggest that rearing on a textured NOX plate was not sufficient to punish operant responding in CFA- and PTX-treated rats despite the presence of sustained mechanical hypersensitivity. The rapid recovery of operant responding could not be attributed to latent sensitization, KOR downregulation, or behavioral tolerance. These results extend the range of conditions under which putative chronic pain manipulations produce weak evidence for depression of operant responding as a sign of the “affective/motivational” component of pain in rats.

Human Intravenous Immunoglobulin Alleviates Neuropathic Symptoms in a Rat Model of Paclitaxel-Induced Peripheral Neurotoxicity.

The onset of chemotherapy-induced peripheral neurotoxicity (CIPN) is a leading cause of the dose reduction or discontinuation of cancer treatment due to sensory symptoms. Paclitaxel (PTX) can cause painful peripheral neuropathy, with a negative impact on cancer survivors’ quality of life. While recent studies have shown that neuroinflammation is involved in PTX-induced peripheral neurotoxicity (PIPN), the pathophysiology of this disabling side effect remains largely unclear and no effective therapies are available. Therefore, here we investigated the effects of human intravenous immunoglobulin (IVIg) on a PIPN rat model. PTX-treated rats showed mechanical allodynia and neurophysiological alterations consistent with a severe sensory axonal polyneuropathy. In addition, morphological evaluation showed a reduction of intra-epidermal nerve fiber (IENF) density and evidenced axonopathy with macrophage infiltration, which was more prominent in the distal segment of caudal nerves. Three weeks after the last PTX injection, mechanical allodynia was still present in PTX-treated rats, while the full recovery in the group of animals co-treated with IVIg was observed. At the pathological level, this behavioral result was paralleled by prevention of the reduction in IENF density induced by PTX in IVIg co-treated rats. These results suggest that the immunomodulating effect of IVIg co-treatment can alleviate PIPN neurotoxic manifestations, probably through a partial reduction of neuroinflammation.

IMMUNOMODULATION AND ANTIGENOTOXIC EFFECTS OF PROPOLIS IN PACLITAXEL-TREATED RATS

The current study aimed to investigate the potential immunomodulatory and antigenotoxic impacts of propolis in paclitaxel (PTX)-treated rats. Twenty four male Sprague Dawley rats were used in the present study and randomly/equally divided into four groups; control group, PTX group that was intraperitoneally injected with 5 mg/kg PTX (once/week), propolis group that received orally/daily 50 mg/kg propolis, and the last group received both PTX and propolis. All treatments were given for four weeks. The results showed a significant upregulation in the nuclear factor kappa B (Nf-κb) gene expression in the spleen of PTX group, as compared with the control group. In addition, substantial increases in tumor necrosis factor-α, interferon-γ, C-X-C motif ligand 10, CC motif ligand 2, and interleukin (IL)-4 concentrations, while a significant reduction in IL-10 concentration, were detected in the spleen of PTX group compared with the control group. Moreover, PTX resulted in a significant elevation in the frequency of micronucleated polychromatic erythrocytes in bone marrow cells compared with the control group. On the other hand, the treatment with propolis alone did not significantly affect all of these parameters, as compared with the control group. However, propolis decreased significantly all recorded side effects in the PTX-treated rats. In conclusion, propolis can be used as an adjunct with PTX to modulate the cytokines and chemokines release of splenic immune cells, as well as to counteract the genotoxic effect of PTX on bone marrow cells, through downregulating the splenic Nf-κb gene expression, and reducing the bone marrow micronuclei formation, respectively

IMMUNOMODULATION AND ANTIGENOTOXIC EFFECTS OF PROPOLIS IN PACLITAXEL-TREATED RATS

The current study aimed to investigate the potential immunomodulatory and antigenotoxic impacts of propolis in paclitaxel (PTX)-treated rats. Twenty four male Sprague Dawley rats were used in the present study and randomly/equally divided into four groups; control group, PTX group that was intraperitoneally injected with 5 mg/kg PTX (once/week), propolis group that received orally/daily 50 mg/kg propolis, and the last group received both PTX and propolis. All treatments were given for four weeks. The results showed a significant upregulation in the nuclear factor kappa B (Nf-κb) gene expression in the spleen of PTX group, as compared with the control group. In addition, substantial increases in tumor necrosis factor-α, interferon-γ, C-X-C motif ligand 10, CC motif ligand 2, and interleukin (IL)-4 concentrations, while a significant reduction in IL-10 concentration, were detected in the spleen of PTX group compared with the control group. Moreover, PTX resulted in a significant elevation in the frequency of micronucleated polychromatic erythrocytes in bone marrow cells compared with the control group. On the other hand, the treatment with propolis alone did not significantly affect all of these parameters, as compared with the control group. However, propolis decreased significantly all recorded side effects in the PTX-treated rats. In conclusion, propolis can be used as an adjunct with PTX to modulate the cytokines and chemokines release of splenic immune cells, as well as to counteract the genotoxic effect of PTX on bone marrow cells, through downregulating the splenic Nf-κb gene expression, and reducing the bone marrow micronuclei formation, respectively

Polyester Nanoparticle Encapsulation Mitigates Paclitaxel-Induced Peripheral Neuropathy.

Chemotherapy utilizing cytotoxic drugs, such as paclitaxel (PTX), is still a commonly used therapeutic approach to treat both localized and metastasized cancers. Unlike traditional regimens in which PTX is administered at the maximum tolerated dose, alternative regimens like metronomic dosing are beneficial by administering PTX more frequently and in much lower doses exploiting antiangiogenic and immunomodulatory effects. However, PTX-induced peripheral neuropathy and lack of patient compliant dosage forms of PTX are major roadblocks for the successful implementation of metronomic regimens. Because of the success of polyester nanoparticle drug delivery, we explored the potential of nanoparticle-encapsulated paclitaxel (nPTX) in alleviating peripheral neuropathy using a rat model. Rats were injected intraperitoneally with 2 mg/kg body weight of PTX or nPTX on four alternate days, and neuropathic pain and neuronal damage were characterized using behavioral assessments, histology, and immunohistochemistry. The reduction in tactile and nociceptive pressure thresholds was significantly less in nPTX-treated rats than in PTX-treated rats over a 16-day study period. Histological analysis showed that the degree of dorsal root ganglion (DRG) degeneration and reduction in motor neurons in the spinal cord was significantly lower in the nPTX group than the PTX group. Further, immunofluorescence data reveals that nPTX-treated rats had an increased density of a neuronal marker, β-tubulin-III, reduced TUNEL positive cells, and increased high molecular weight neurofilament in the spinal cord, DRG, and sciatic nerves compared with PTX-treated rats. Therefore, this work has important implications in improving risk-benefit profile of PTX, paving the way for metronomic regimens.

Protective effect of pentoxifylline on oxidative renal cell injury associated with renal crystal formation in a hyperoxaluric rat model.

The aim of the study is to investigate the effects of pentoxifylline (PTX) on the renal tubular cell injury and stone formation in a hyperoxaluric rat model induced by ethylene glycol and its possible underlying mechanisms. The study was performed with 30 male Wistar rats and randomized into three groups of teen. The sham-control (group 1) received only drinking water orally. The EG/untreated (group 2) received drinking water containing 0.75% EG for 4weeks orally. The EG/PTX treated (group 3) received drinking water containing 0.75% EG for 4weeks orally and PTX. Urine and blood were collected to determine some parameters. The kidneys were also removed for histological examination. Serum and urinary parameters were significantly improved in the EG/PTX treated. In the EG/PTX-treated group, the MDA, TOS and MPO activity reduced and the TAS, SOD, CAT and GSH-Px activities were increased markedly compared with the group 2. In urine of the group 2 rats, a large number of CaOx crystals were displayed and most tubules that contained crystals were dilated and showed degeneration, necrosis, and desquamation of the lining epithelium. Only few CaOx crystals were r in EG/PTX-treated animal's urine. Mild tissue damage was observed in PTX-treated rats. iNOS expression was significantly elevated in the group 2. In contrast, in the EG/PTX-treated group, eNOS expression in renal tubular epithelial cells was increased. Current study indicates that PTX may partially reduce renal tubular injury resulting from hyperoxaluria-induced oxidative and nitrosative stress.

The effects of pentoxifylline adminstration on fracture healing in a postmenopausal osteoporotic rat model

Previous studies report positive effects of pentoxifylline (PTX) alone or in combination with other drugs on some pathologic bone diseases as well as an ability to accelerate osteogensis and fracture healing in both animal models and human patients. The aim of this present study was to evaluate the effects of PTX administration on Hounsfield unit and bone strength at catabolic response (bone resorbing) of a fracture in an experimental rat model of ovariectomy induced osteoporosis (OVX-D). Thirty adult female rats were divided into groups as follows: 1 (OVX, control, no treatment); 2 (OVX, sham: daily distilled water); 3 (OVX, daily alendronate: 3 mg/kg); 4 (OVX, twice daily 100 mg/kg PTX) and 5 (OVX, PTX+alenderonate). OVX was induced by bilateral ovariectomy in all rats. A complete standardized osteotomy of the right femur was made after 3.5 months. PTX and alendronate treatments were performed for eight weeks. Then, rats were euthanized and had its right femur subjected to computerized tomography scanning for measuring Hounsfield unit; eventually, the samples were sent for a three point bending test for evaluation of the bone strength. Administration of PTX with 200 mg/kg and alendronate alone and in combination showed no significant alteration in Hounsfield unit and biomechanical properties of repairing callus of the complete osteotomy compared with the control group. Results showed increased bending stiffness and stress high load mean values of repairing complete osteotomy in PTX-treated rats compared to the control OVX-D.

Neuroprotective effects of p-tyrosol after the global cerebral ischemia in rats

BackgroundSalidroside is a biologically active compound derived from Rhodiola rosea L. Studies showed that salidroside after i.v. injection is extensively metabolized to p-tyrosol and only trace amounts of salidroside are found in the brain tissue. ObjectiveThe aim of the study was to investigate the neuroprotective effects of p-tyrosol in the global cerebral ischemia-reperfusion (GCI) model. Study designA total of 103 Wistar rats were assigned to groups of sham-operated (n=10), control (n=42), p-tyrosol-treated (n=36), and pentoxifylline-treated (n=15) animals. The rats of control, p-tyrosol-treated, and pentoxifylline-treated groups received intravenously 0.9% NaCl solution, 2% solution of p-tyrosol in doses of 5mg/kg, 10mg/kg, and 20mg/kg, and pentoxifylline in a dose of 100mg/kg, respectively, daily for 5 days. Rats were examined at days 1, 3, and 5 after GCI. After evaluation of neurological deficit, animals were euthanized for morphological and biochemical characterization. MethodsRats of control, p-tyrosol-treated, and pentoxifylline-treated groups were exposed to three-vessel model of GCI. Neurological deficit, numeric density of neurons in hippocampal CA1 region, and percentage of neurons with focal and total chromatolysis were studied. Biochemical study assessed contents of conjugated dienes and fluorescent products in brain homogenate. ResultsIn control group, only 50.0% of rats survived by day 5 after the GCI; 38.1% of survived animals had severe neurologic deficit. In brain tissue of PTX-treated rats, the levels of diene conjugates and fluorescent products were 79% and 73%, respectivley, at day 5 compared with control. Differences in diene conjugates were statistically significant compared with control. The survival rate of animals treated with 20mg/kg p-tyrosol was 82.3% at day 5 after GCI. In p-tyrosol-treated GCI rats, the numeric density of neurons in the hippocampal CA1 region was higher by 31% compared with control. The percentage of neurons with focal and total chromatolysis decreased by 27% and 43%, respectively. At day 5 after GCI, the levels of conjugated dienes and fluorescent products were significantly lower (by 37% and 45%, respectively) in group of animals treated with 20mg/kg p-tyrosol compared with control. Moderate neuroprotective effects of 5mg/kg p-tyrosol administration were documented only at day 5 after GCI. In case of 10mg/kg p-tyrosol administration, neuroprotection was documented sooner: at day 1 or 3 after GCI. However, administration of 5 and 10mg/kg p-tyrosol did not affect animal survival. ConclusionCourse administration of intravenous p-tyrosol in a dose of 20mg/kg increased survival, reduced neurological deficit after GCI, attenuated neuronal damage in the hippocampus, and attenuated lipid peroxidation in brain tissue in animals subject to GCI with reperfusion.

Abstract P1-15-07: The neuroprotective aminopropyl carbazole, P7C3-A20, prevents paclitaxel-induced pain

Abstract Paclitaxel (PTX), a microtubule-targeting anticancer agent, produces a debilitating peripheral neuropathy that is accompanied by neuropathic pain. Currently, there are only marginally effective therapeutic interventions available. Consequently, patients are forced to reduce or discontinue life-saving chemotherapy to cope with the pain. Recently, a newly identified agent, P7C3-A20, was found to be protective in several models of neurodegeneration, including Parkinson's disease and traumatic brain injury. Given that PTX triggers progressive degeneration of peripheral afferent neurons, this study was performed to evaluate the potential neuroprotective efficacy of P7C3-A20 in rodent models of PTX-induced peripheral neuropathy. P7C3-A20 (10 mg/kg) or vehicle (Cremophor EL/DMSO/5% Dextrose; 1:1:3) was administered intraperitoneally (i.p.) to Sprague-Dawley rats (250-300 g) everyday over a 28-day experimental paradigm. Following two days of treatment with P7C3-A20 or vehicle, rats also received 3 injections of PTX (11.7 mg/kg, i.p.) or vehicle (Cremophor EL/DMSO/5% Dextrose; i.p.) administered every other day. Treatment with P7C3-A20 did not alter body weights or leukocyte counts in control or PTX-treated rats. PTX treatment increased sensitivity to mechanical and cold stimulation (allodynia) of the hindpaw, evidence of peripheral neuropathy. Notably, P7C3-A20 treatment prevented the development of allodynia in response to PTX. Immunohistochemical analysis of paw biopsies indicated that P7C3-A20 prevented PTX-mediated degeneration of terminal nerve endings. Collectively, these data suggest that P7C3-A30 prevented the neurotoxic effects of PTX on peripheral sensory neurons. A xenograft tumor model of triple negative breast cancer was then used to determine whether P7C3-A20 diminished the antitumoral efficacy of PTX. MDA-MB-231 tumors were bilaterally implanted into flanks of female athymic nude mice and allowed to grow for 3 weeks prior to treatment. Using the same treatment protocol as the rats, tumor-bearing mice received daily treatment with P7C3-A20 (20 mg/kg, i.p.) or vehicle for 16 days. P7C3-A20 did not alter PTX-mediated inhibition of tumor growth. Furthermore, P7C3-A20 prevented PTX-induced mechanical allodynia in the mice. Taken together, this work indicates that P7C3-A20 prevents PTX-induced neuropathic damage without diminishing its antitumoral efficacy. P7C3-A20 may be an exciting new candidate to prevent peripheral neuropathy in patients undergoing cancer treatment with PTX. Citation Format: LoCoco PM, Risinger AL, Mooberry SL, Berg KA, Clarke WP. The neuroprotective aminopropyl carbazole, P7C3-A20, prevents paclitaxel-induced pain. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-15-07.

Abstract P3-12-06: Subpopulations of peripheral sensory neurons are differentially sensitive to the microtubule-targeting agent, paclitaxel

Abstract Paclitaxel (PTX), a microtubule-targeting anticancer agent, produces a debilitating peripheral neuropathy that is accompanied by neuropathic pain. Patients develop localized pain in their distal extremities, and more frequently complain of increased sensitivity to cold rather than hot temperatures. These clinical observations led us to hypothesize that PTX differentially damages subpopulations of peripheral sensory neurons. The purpose of this study was to evaluate the effects of PTX on bradykinin (BK) and eicosanoid (EP) receptor-expressing sensory neurons. PTX (11.7 mg/kg) or vehicle (Cremophor EL/EtOH/PBS; 1:1:6) was administered intraperitoneally to Sprague-Dawley rats (250-300 g) every other day for 5 days. Following treatment, PTX-treated rats exhibited increased sensitivity to cold and mechanical stimuli, but decreased sensitivity to heat stimulation as compared to vehicle-treated rats. Intraplantar (i.pl.) injection with BK (5 μg) produced a prolonged allodynia to cold, but a diminished allodynia to heat in PTX-treated rats. Interestingly, following treatment with PGE2 (0.3 μg, i.pl.), PTX-treated rats displayed no statistically significant differences in the allodynia to cold or heat compared to vehicle-treated rats. Efficacy of BK (1 pM - 1 μM) to stimulate inositol phosphate (IP) production in cultured primary sensory neurons from PTX-treated rats was significantly reduced. By contrast, stimulation of cAMP production by PGE2 (0.01 pM - 100 nM) was unaffected by PTX treatment. Preliminary mechanistic studies treating naïve cultures of rat primary sensory neurons directly with PTX (5 nM, 0 - 48 hr) yielded a time-dependent, tri-phasic effect on BK-stimulated IP accumulation and reduced PGE2-stimulated cAMP production, suggesting PTX differentially disrupts BK- and PGE2-mediated signaling. Collectively, these data provide evidence that subpopulations of peripheral sensory neurons are differentially sensitive to PTX. Understanding why particular subclasses are less sensitive, or resistant, to damage could reveal novel approaches to protect sensory neurons from chemotoxicity and ultimately prevent chemotherapy-induced peripheral neuropathy. Supported by USPS grant #8ULITR000149, Greehey Fellowship, and Translational Science Training Across Disciplines Program at UTHSCSA The project described was supported by Award Number 8UL1TR000149 from the National Center for Advancing Translational Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Advancing Translational Sciences. Citation Format: Peter M LoCoco, Teresa C Chavera, Raehannah J Jamshidi, Susan L Mooberry, Kelly A Berg, William P Clarke. Subpopulations of peripheral sensory neurons are differentially sensitive to the microtubule-targeting agent, paclitaxel [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-12-06.

Effects of Late Administration of Pentoxifylline and Tocotrienols in an Image-Guided Rat Model of Localized Heart Irradiation

Radiation-induced heart disease (RIHD) is a long-term side effect of radiotherapy of intrathoracic, chest wall and breast tumors when radiation fields encompass all or part of the heart. Previous studies have shown that pentoxifylline (PTX) in combination with α-tocopherol reduced manifestations of RIHD in rat models of local heart irradiation. The relative contribution of PTX and α-tocopherol to these beneficial effects are not known. This study examined the effects of PTX alone or in combination with tocotrienols, forms of vitamin E with potential potent radiation mitigation properties. Rats received localized X-irradiation of the heart with an image-guided irradiation technique. At 3 months after irradiation rats received oral treatment with vehicle, PTX, or PTX in combination with a tocotrienol-enriched formulation. At 6 months after irradiation, PTX-treated rats showed arrhythmia in 5 out of 14 animals. PTX alone or in combination with tocotrienols did not alter cardiac radiation fibrosis, left ventricular protein expression of the endothelial markers von Willebrand factor and neuregulin-1, or phosphorylation of the signal mediators Akt, Erk1/2, or PKCα. On the other hand, tocotrienols reduced cardiac numbers of mast cells and macrophages, but enhanced the expression of tissue factor. While this new rat model of localized heart irradiation does not support the use of PTX alone, the effects of tocotrienols on chronic manifestations of RIHD deserve further investigation.

Fibrin-Induced epithelial-to-mesenchymal transition of peritoneal mesothelial cells as a mechanism of peritoneal fibrosis: effects of pentoxifylline.

Excessive fibrin deposition in the peritoneum is thought to be involved in the development of encapsulating peritoneal sclerosis (EPS), an important cause of morbidity and mortality in peritoneal dialysis patients. We investigated fibrin-induced epithelial-to-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) as a possible mechanism of fibrin involvement in EPS. In vitro, fibrin overlay of PMCs altered their morphology; increased α-smooth muscle actin, fibronectin, fibroblast specific protein-1, and αvβ3 integrin expression; and decreased cytokeratin 18 and E-cadherin expression. Fibrin overlay also increased focal adhesion kinase and Src kinase phosphorylation. Fibrin-induced changes were inhibited by treating the cells with αvβ3 integrin antibody or pentoxifylline (PTX). In a rat model, intraperitoneal injection of Staphylococcus aureus and fibrinogen induced severe EPS features, which were attenuated by PTX treatment. PTX-treated rats also showed preserved peritoneal ultrafiltration function and lower concentrations of cytokines than the untreated rats. S. aureus- and fibrinogen-injected rats had higher percentage of cytokeratin-positive cells in the omentum fibrotic tissue than controls; this was also reduced by PTX treatment. Our results suggest that fibrin induces EMT of PMCs by engaging αvβ3 integrin and activating associated kinases. Our EPS animal model showed that fibrin-induced EMT was involved in the pathogenesis of peritoneal fibrosis and was inhibited by PTX.

Ultra-low-dose naloxone enhances the antinociceptive effect of morphine in PTX-treated rats: Regulation on global histone methylation

ObjectiveEpigenetic reprogramming may have a possible role in neuropathic pain development; the present study examined the global patterns of lysine histone modification. In this serial study we analyzed the levels of histone 3 lysine 4 monomethylation, histone 3 lysine 4 dimethylation, and histone 3 lysine 9 trimethylation in pertussis toxin (PTX)-induced thermal hyperalgesic rat spinal cords. MethodsMale Wistar rats implanted with an intrathecal catheter received a single intrathecal PTX (1μg in 5μl saline) injection. Four days later, they were randomly assigned to receive either a single injection of saline, or ultra-low-dose naloxone (15ng in 5μl saline), followed by morphine (10μg in 5μl saline) injection 30 minutes later. ResultsThe results showed that PTX injection induced thermal hyperalgesia and significant increase of global histone methylation in the spinal cords. Intrathecal morphine alone did not affect the thermal hyperalgesia and global histone methylation. In contrast, intrathecal administration of ultra-low-dose naloxone plus morphine significantly attenuated the PTX-induced thermal hyperalgesia and down-regulated the global histone methylation. ConclusionThe results suggest that ultra-low-dose naloxone might be clinical valuable for neuropathic pain management via regulating global histone modification.

Ultra-low dose naloxone restores the antinocicepitve effect of morphine in PTX-treated rats: Association of IL-10 upregulation in the spinal cord

AimsUltra-low dose naloxone has been shown to restore the antinociceptive effect of morphine in pertussis toxin (PTX)-treated rats by suppressing spinal microglia activation and inhibiting inflammatory cytokine expression. This study was further investigated the mechanism by which ultra-low dose naloxone promotes analgesia in pertussis toxin-treated rats. Main methodsMale Wistar rats were implanted with an intrathecal (i.t.) catheter and injected either saline or PTX (1μg). Four days later, rats randomly received either saline, or ultra-low dose naloxone, or recombinant rat interleukin-10 (rrIL-10) (1μg) injection followed by saline or morphine (10μg) 30min later. In some experiments, mouse anti-rat IL-10 antibody (10μg) was injected intrathecally into PTX injected rats daily on days 4, 5, 6, and 7. On day 7, ultra-low dose naloxone was given 1h after antibody injection with or without subsequent morphine injection. Key findingsPTX injection induced notable thermal hyperalgesia and mechanical allodynia. Injection of ultra-low dose naloxone preserved the antinociceptive effect of morphine in PTX-treated rats and associated an increasing of IL-10 protein expression. Intrathecal injection rrIL-10 alone or in combination with morphine, not only reversed mechanical allodynia but also partially restored the antinociceptive effect of morphine; injection of anti-rat IL-10 antibody attenuated the effect of morphine plus ultra-low dose naloxone on mechanical allodynia and completely inhibited the antinociceptive effect of morphine. SignificanceThese results indicate that intrathecal ultra-low dose naloxone induces IL-10 expression in spinal neuron and microglia, which suppresses PTX-induced neuroinflammation and restores the antinociceptive effect of morphine.

The Inhibitory G Protein G<sub>i</sub> Identified as Pertussis Toxin-Catalyzed ADP-Ribosylation

Pertussis toxin (PTX) produced by Bordetella pertussis was first introduced by Ui and his colleagues in research on signal transduction under the name islet-activating protein in 1979, when the mechanism of toxin-induced stimulation of insulin release from pancreatic islets was reported in the rat. The stimulatory effect of PTX in vivo results from the blockage of α(2)-adrenergic receptor-mediated inhibition of insulin release. The receptor-induced inhibition of cAMP formation was also abolished in pancreatic islets isolated from PTX-treated rats, suggesting that the toxin caused uncoupling of adenylyl cyclase inhibition from receptor stimulation. The action of PTX on isolated membranes required a cytosolic factor, nicotinamide adenine dinucleotide (NAD), and the uncoupling induced by PTX was shown to be due to the toxin-catalyzed ADP-ribosylation of a 41-kDa protein with NAD as another substrate. The 41-kDa PTX substrate was soon identified and purified as the α-subunit of the inhibitory G protein that transmits an inhibitory signal from membrane receptors to adenylyl cyclase. After demonstration of the molecular mechanism of PTX, the toxin was widely utilized as a probe for identifying and analyzing major αβγ-trimeric G proteins. Thus, PTX-sensitive G proteins appeared to carry positive and negative signals from many membrane receptors to a variety of effectors other than adenylyl cyclase.

Ultra-low dose naloxone restores the antinociceptive effect of morphine in pertussis toxin-treated rats by reversing the coupling of μ-opioid receptors from Gs-protein to coupling to Gi-protein

Pertussis toxin (PTX) treatment results in ADP-ribosylation of Gi-protein and thus in disruption of μ-opioid receptor signal transduction and loss of the antinociceptive effect of morphine. We have previously demonstrated that pretreatment with ultra-low dose naloxone preserves the antinociceptive effect of morphine in PTX-treated rats. The present study further examined the effect of ultra-low dose naloxone on μ-opioid receptor signaling in PTX-treated rats and the underlying mechanism. Male Wistar rats implanted with an intrathecal catheter received an intrathecal injection of saline or PTX (1 μg in 5 μl of saline), then, 4 days later, were pretreated by intrathecal injection with either saline or ultra-low dose naloxone (15 ng in 5 μl of saline), followed, 30 min later, by saline or morphine (10 μg in 5 μl of saline). Four days after PTX injection, thermal hyperalgesia was observed, together with increased coupling of excitatory Gs-protein to μ-opioid receptors in the spinal cord. Ultra-low dose naloxone pretreatment preserved the antinociceptive effect of morphine, and this effect was completely blocked by the μ-opioid receptor antagonist CTOP, but not by the κ-opioid receptor antagonist nor-BNI or the δ-opioid receptor antagonist naltrindole. Moreover, a co-immunoprecipitation study showed that ultra-low dose naloxone restored μ-opioid receptor/Gi-protein coupling and inhibited the PTX-induced μ-opioid receptor/Gs-protein coupling. In addition to the anti-neuroinflammatory effect and glutamate transporter modulation previously observed in PTX-treated rats, the re-establishment of μ-opioid receptor Gi/Go-protein coupling is involved in the restoration of the antinociceptive effect of morphine by ultra-low dose naloxone pretreatment by normalizing the balance between the excitatory and inhibitory signaling pathways. These results show that ultra-low dose naloxone preserves the antinociceptive effect of morphine, suppresses spinal neuroinflammation, and reduces PTX-elevated excitatory Gs-coupled opioid receptors in PTX-treated rats. We suggest that ultra-low dose naloxone might be clinically valuable in pain management.

N-Methyl- d-aspartate receptor antagonist d-AP5 prevents pertussis toxin-induced alterations in rat spinal cords by inhibiting increase in concentrations of spinal CSF excitatory amino acids and downregulation of glutamate transporters

Recently, we found that intrathecal (i.t.) pertussis toxin (PTX) injection produces thermal hyperalgesia and is associated with increasing concentrations of excitatory amino acids (EAAs) in spinal cerebrospinal fluid (CSF) dialysates; a reduction in the antinociceptive effects of morphine and glutamate transporters (GTs) was also observed. The reduction in the morphine-induced analgesic effects is directly related to increased extracellular EAA levels, which are maintained by GTs at physiological levels. In this study, we aimed to examine the role of GT isoforms in thermal hyperalgesia, determine the EAA concentrations in CSF dialysates, and elucidate the role of N-methyl- d-aspartate (NMDA) receptors in PTX-induced reduction in the antinociceptive effects of morphine. Two i.t. catheters and one microdialysis probe were inserted into male Wistar rats: one catheter was used for PTX (1 μg) and morphine (10 μg) injection and the other was connected to an osmotic pump for NMDA receptor antagonist d-2-amino-5-phosphonopentanoic acid ( d-AP5; 2 μg/h for 4 days) continuous infusion. The microdialysis probe was used to collect CSF dialysates for EAA measurements by high-performance liquid chromatography. Intrathecal morphine failed to produce antinociceptive effects in PTX-treated rats, and d-AP5 coinfusion prevented the PTX-induced reduction in the antinociceptive effect and associated downregulation of the GTs. We conclude that NMDA receptor suppression inhibits EAA excitation and reduces the morphine-induced antinociception in PTX-treated rats.

Ultra-low dose naloxone restores the antinociceptive effect of morphine in pertussis toxin–treated rats and prevents glutamate transporter downregulation by suppressing the p38 mitogen-activated protein kinase signaling pathway

We previously demonstrated that ultra-low dose naloxone restores the antinociceptive effect of morphine in rats with pertussis toxin (PTX)–induced thermal hyperalgesia by reversing the downregulation of glutamate transporter (GT) expression and suppressing spinal neuroinflammation. In the present study, we examined the underlying mechanisms of this anti-inflammatory effect in PTX-treated rats, particularly on the expression of GTs. Male Wistar rats were implanted with an intrathecal catheter and, in some cases, with a microdialysis probe. All rats were injected intrathecally with saline (5 μl) or PTX (1 μg), then, 4 days later, were randomly assigned to receive a single injection of saline, ultra-low dose naloxone (15 ng), or the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (5 μg), followed by morphine injection (10 μg) 30 min later. Our results showed that PTX injection induced activation of microglia and a significant increase in P-p38 MAPK expression in the spinal cord. Ultra-low dose naloxone plus morphine significantly inhibited the effect of PTX on P-p38 MAPK expression in the spinal cord, while the p38 MAPK inhibitor SB203580 attenuated the PTX-induced mechanical allodynia, thermal hyperalgesia, increase in spinal cerebrospinal fluid excitatory amino acids, and downregulation of GTs. These results show that the restoration of the antinociceptive effect of morphine and GT expression in PTX-treated rats by ultra-low dose naloxone involves suppression of the p38 MAPK signal transduction cascade.