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Poly(d,l-lactide)/polyethylene glycol (PLA/PEG) micro/nanofibers loaded with paclitaxel (PTX, 10 wt%) were prepared by needless electrospinning technology, which allows large scale production for real medicinal practice. The fiber structure and properties were investigated by several methods including scanning electron microscopy, nitrogen adsorption/desorption isotherm measurements, differential scanning calorimetry, and X-ray diffraction measurements to examine their morphology (fiber diameter distribution, specific surface area, and total pore volume), composition, drug-loading efficiency, and physical state. An HPLC-UV method was optimized and validated to quantify in vitro PTX release into PBS. The results showed that the addition of PEG into PLA fibers promoted the release of higher amounts of hydrophobic PTX over prolonged time periods compared to fibers without PEG. An in vitro cell assay demonstrated the biocompatibility of PLA/PEG fibrous materials and showed significant cytotoxicity of PTX-loaded PLA/PEG fibers against a human fibrosarcoma HT1080 cell line. The chick chorioallantoic membrane assay proved that PTX-loaded fibers exhibited antiangiogenic activity, with a pronounced effect in the case of the PEG-containing fibers. In vivo evaluation of PTX-loaded PLA/PEG fibers in a human fibrosarcoma recurrence model showed statistically significant inhibition in tumor incidence and growth after primary tumor resection compared to other treatment groups., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cachectic rheumatoid arthritis, the less frequent form of the disease, is associated with loss of fat mass and often more severe course of the disease. Its experimental model represents rat adjuvant arthritis (AA) characterized by edema, lack of appetite, sharp body weight and fat loss. As individual fat depots display functional differences, here we studied lipolytic activity and sensitivity to lipolytic stimuli of nodeless epididymal fat (eWAT) and perinodal mesenteric fat (mWAT) depots at the peak of AA. We also examined changes in catecholamine and cytokine levels involved in lipolysis in plasma and/or isolated adipocytes from both WATs to identify the contribution of local, adipocyte-based processes and/or systemic events to adiposity loss in cachectic rheumatoid arthritis. AA was induced to male Lewis rats by complete Freund's adjuvant. Groups of ad libitum-fed and pair-fed controls were used to distinguish the effects of food restriction from inflammation-induced cachexia. Adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and its phosphorylated form (pHSL) were analyzed by western blot. CRP and catecholamine levels in plasma or adipocyte lysates were determined using ELISA kits. Cytokine-induced neutrophil chemoattractant-1 (CINC-1/CXCL1), monocyte chemoattractant protein-1 (MCP-1/CCL2), IL-1β, IL-6, IL-10 and leptin in adipocyte lysate were analyzed by quantitative protein microarray. Plasma glycerol and FFA were measured spectrophotometrically. AA rats developed severe cachexia, with lower adiposity in mWAT compared to normal and pair-fed controls, whereas in eWAT the adiposity was similarly reduced in AA and pair-fed groups. ATGL levels in both WATs were not affected by AA or pair feeding. AA upregulated levels of HSL, pHSL and pHSL/HSL ratio in mWAT, whereas none of these parameters has changed in eWAT of AA rats or in either WATs of pair-fed rats. In AA rats plasma glycerol was elevated, whereas FFA concentration was reduced. Plasma norepinephrine and epinephrine were increased in AA compared with both groups of controls. In eWAT adipocytes, AA but not pair feeding, upregulated norepinephrine levels. In mWAT adipocytes, AA rats showed higher epinephrine levels than pair-fed controls. Leptin levels in both WATs were depleted in AA animals in accordance with body weight loss. None of the measured cytokines in eWAT and mWAT was enhanced. Our results demonstrate augmented lipolytic activity in mWAT and not eWAT during cachectic arthritis. The adipocyte-derived cytokines do not seem to contribute to activated lipolysis. We first demonstrated enhanced presence of norepinephrine in perinodal adipocytes that may contribute to the regulation of local lipolytic activity by auto/paracrine fashion and thus provide independent fuel supply to activated lymph nodes.

Reactive oxygen species play an important role both in physiological and pathophysiological reactions. The aim of this study was to determine the role of free radicals and antioxidants in the development of visceral pain. Visceral pain was produced by colorectal distension (CRD) in adult rats. CRD was caused by insertion of a lubricated latex balloon into the descending colon and rectum followed by inflation to 80mm Hg for 10min. During CRD, visceral pain was rated on 0-3.5 point scale. Oxidative stress was determined indirectly by measurement of free radical scavenging enzymes (glutathione peroxidase (GPx) and superoxide dismutase (SOD)) in the blood, liver and brain. Following CRD we observed (1) all rats expressed signs of visceral pain (overall rating was 1.83), (2) SOD and GPx levels were increased in the liver and blood, and decreased in the brain samples and (3) administration of the antioxidant Trolox, a water-soluble derivate of vitamin E, prior to CRD, prevented SOD and GPx changes in the liver, blood and brain, but did not affect pain scores. It was concluded, that CRD as a model of visceral pain, increases oxidative stress in animals, which could be prevented by prior administration of antioxidants; however, antioxidants did not attenuate signs of visceral pain caused by CRD., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

While the effect of cortex stimulation on pain control is widely accepted, its physiological basis remains poorly understood. We chose an animal model of pain to study the influence of sensorimotor cortex stimulation on tooth pulp stimulation evoked potentials (TPEPs). Fifteen awake rats implanted with tooth pulp, cerebral cortex, and digastric muscle electrodes were divided into three groups, receiving 60 Hz, 40 Hz and no cortical stimulation, respectively. TPEPs were recorded before, one, three and five hours after continuous stimulation. We observed an inverse relationship between TPEP amplitude and latency with increasing tooth pulp stimulation. The amplitudes of the early components of TPEPs increased and their latency decreased with increasing tooth pulp stimulation intensity. Cortical stimulation decreased the amplitude of TPEPs; however, neither the latencies of TPEPs nor the jaw-opening reflex were changed after cortical stimulation. The decrease in amplitude of TPEPs after cortical stimulation may reflect its anti-nociceptive effect.

Background: Baclofen, a specific GABA(B) receptor agonist, is used to treat spasticity and its off-label use includes the treatment of pain. The aim of this study was to show the role of baclofen in acute pain modulation during the early postnatal period., Methods: Baclofen was tested in 2 doses (1 and 5 mg/kg) in 3 age groups (postnatal days 7, 16 and 21) in order to assess its effect on acute nociception and motor co ordination in rat pups. Pain was evaluated by measurement of paw/tail withdrawal latencies, and motor coordination by age-relevant tests., Results: Although baclofen impaired motor function in all age groups, no changes in paw withdrawal latencies were observed. On the other hand, baclofen significantly increased tail withdrawal latencies of all groups., Conclusion: It is concluded that the GABA(B) system is functionally matured in the early postnatal period and plays a similar, but not the same, role as in adulthood.

Cholangiopathies are characterized by the heterogeneous proliferation of different-sized cholangiocytes. Large cholangiocytes proliferate by a cAMP-dependent mechanism. The function of small cholangiocytes may depend on the activation of inositol trisphosphate (IP(3))/Ca(2+)-dependent signaling pathways; however, data supporting this speculation are lacking. Four histamine receptors exist (HRH1, HRH2, HRH3, and HRH4). In several cells: 1) activation of HRH1 increases intracellular Ca(2+) concentration levels; and 2) increased [Ca(2+)](i) levels are coupled with calmodulin-dependent stimulation of calmodulin-dependent protein kinase (CaMK) and activation of cAMP-response element binding protein (CREB). HRH1 agonists modulate small cholangiocyte proliferation by activation of IP(3)/Ca(2+)-dependent CaMK/CREB. We evaluated HRH1 expression in cholangiocytes. Small and large cholangiocytes were stimulated with histamine trifluoromethyl toluidide (HTMT dimaleate; HRH1 agonist) for 24-48 h with/without terfenadine, BAPTA/AM, or W7 before measuring proliferation. Expression of CaMK I, II, and IV was evaluated in small and large cholangiocytes. We measured IP(3), Ca(2+) and cAMP levels, phosphorylation of CaMK I, and activation of CREB (in the absence/presence of W7) in small cholangiocytes treated with HTMT dimaleate. CaMK I knockdown was performed in small cholangiocytes stimulated with HTMT dimaleate before measurement of proliferation and CREB activity. Small and large cholangiocytes express HRH1, CaMK I, and CaMK II. Small (but not large) cholangiocytes proliferate in response to HTMT dimaleate and are blocked by terfenadine (HRH1 antagonist), BAPTA/AM, and W7. In small cholangiocytes, HTMT dimaleate increased IP(3)/Ca(2+) levels, CaMK I phosphorylation, and CREB activity. Gene knockdown of CaMK I ablated the effects of HTMT dimaleate on small cholangiocyte proliferation and CREB activation. The IP(3)/Ca(2+)/CaMK I/CREB pathway is important in the regulation of small cholangiocyte function.

During cholestatic liver diseases, cholangiocytes express neuroendocrine phenotypes and respond to a number of hormones and neuropeptides by paracrine and autocrine mechanisms. We examined whether the neuroendocrine hormone progesterone is produced by and targeted to cholangiocytes, thereby regulating biliary proliferation during cholestasis. Nuclear (PR-A and PR-B) and membrane (PRGMC1, PRGMC2, and mPRalpha) progesterone receptor expression was evaluated in liver sections and cholangiocytes from normal and bile duct ligation (BDL) rats, and NRC cells (normal rat cholangiocyte line). In vivo, normal rats were chronically treated with progesterone for 1 wk, or immediately after BDL, rats were treated with a neutralizing progesterone antibody for 1 wk. Cholangiocyte growth was measured by evaluating the number of bile ducts in liver sections. The expression of the progesterone synthesis pathway was evaluated in liver sections, cholangiocytes and NRC. Progesterone secretion was evaluated in supernatants from normal and BDL cholangiocytes and NRC. In vitro, NRC were stimulated with progesterone and cholangiocyte supernatants in the presence or absence of antiprogesterone antibody. Aminoglutethimide was used to block progesterone synthesis. Cholangiocytes and NRC express the PR-B nuclear receptor and PRGMC1, PRGMC2, and mPRalpha. In vivo, progesterone increased the number of bile ducts of normal rats, whereas antiprogesterone antibody inhibited cholangiocyte growth stimulated by BDL. Normal and BDL cholangiocytes expressed the biosynthetic pathway for and secrete progesterone. In vitro, 1) progesterone increased NRC proliferation; 2) cholangiocyte supernatants increased NRC proliferation, which was partially inhibited by preincubation with antiprogesterone; and 3) inhibition of progesterone steroidogenesis prevented NRC proliferation. In conclusion, progesterone may be an important autocrine/paracrine regulator of cholangiocyte proliferation.

The endocannabinoid system regulates various aspects of hepatic fibrosis; however, nothing is known about its role in regulating cholangiocyte proliferation and function. We evaluated the effects of anandamide (AEA) on cholangiocyte proliferation and explored the effects of AEA on the thioredoxin 1 (TRX1)/redox factor 1 (Ref1)/activator protein-1 (AP-1) pathway. Mice underwent bile duct ligation (BDL) and were infused with AEA for 3 days postsurgery. Proliferation and apoptosis were evaluated in liver sections. Effects of in vitro AEA treatment on cholangiocyte proliferation and apoptosis were studied in purified cholangiocytes. The relative expression of cannabinoid receptors was also assessed in liver sections and cholangiocytes. mRNA expression of the cannabinoid receptors Cb1 and VR1 was decreased after BDL, whereas there was an upregulation of Cb2 mRNA. AEA decreased cholangiocyte growth and induced accumulation of reactive oxygen species, upregulation of TRX1, Ref1, c-Fos, and c-Jun expression, increased nuclear localization of TRX1, and increased AP-1 transcriptional activity. Specific knockdown of TRX1 or Ref1 expression ablated the AP-1 transcriptional activity and AEA-induced cell death but not expression of c-Fos and c-Jun. Knockdown of c-Fos and c-Jun expression also ablated AEA-induced apoptosis. We conclude that AEA suppresses cholangiocyte proliferation during cholestasis via a Cb2-dependent mechanism. Modulation of the endocannabinoid system may be important in the treatment of cholangiopathies.

Background: Prolactin promotes proliferation of several cells. Prolactin receptor exists as two isoforms: long and short, which activate different transduction pathways including the Ca2+-dependent PKC-signaling. No information exists on the role of prolactin in the regulation of the growth of female cholangiocytes. The rationale for using cholangiocytes from female rats is based on the fact that women are preferentially affected by specific cholangiopathies including primary biliary cirrhosis. We propose to evaluate the role and mechanisms of action by which prolactin regulates the growth of female cholangiocytes., Results: Normal cholangiocytes express both isoforms (long and short) of prolactin receptors, whose expression increased following BDL. The administration of prolactin to normal female rats increased cholangiocyte proliferation. In purified normal female cholangiocytes, prolactin stimulated cholangiocyte proliferation, which was associated with increased [Ca2+]i levels and PKCbeta-I phosphorylation but decreased PKCalpha phosphorylation. Administration of an anti-prolactin antibody to BDL female rats decreased cholangiocyte proliferation. Normal female cholangiocytes express and secrete prolactin, which was increased in BDL rats. The data show that prolactin stimulates normal cholangiocyte growth by an autocrine mechanism involving phosphorylation of PKCbeta-I and dephosphorylation of PKCalpha., Conclusion: We suggest that in female rats: (i) prolactin has a trophic effect on the growth of normal cholangiocytes by phosphorylation of PKCbeta-I and dephosphorylation of PKCalpha; and (iii) cholangiocytes express and secrete prolactin, which by an autocrine mechanism participate in regulation of cholangiocyte proliferation. Prolactin may be an important therapeutic approach for the management of cholangiopathies affecting female patients.

Cholangiocarcinomas are devastating cancers of biliary origin with limited treatment options. Modulation of the endocannabinoid system is being targeted to develop possible therapeutic strategies for a number of cancers; therefore, we evaluated the effects of the two major endocannabinoids, anandamide and 2-arachidonylglycerol, on numerous cholangiocarcinoma cell lines. Although anandamide was antiproliferative and proapoptotic, 2-arachidonylglycerol stimulated cholangiocarcinoma cell growth. Specific inhibitors for each of the cannabinoid receptors did not prevent either of these effects nor did pretreatment with pertussis toxin, a G(i/o) protein inhibitor, suggesting that anandamide and 2-arachidonylglycerol did not exert their diametric effects through any known cannabinoid receptor or through any other G(i/o) protein-coupled receptor. Using the lipid raft disruptors methyl-beta-cyclodextrin and filipin, we demonstrated that anandamide, but not 2-arachidonylglycerol, requires lipid raft-mediated events to inhibit cellular proliferation. Closer inspection of the lipid raft structures within the cell membrane revealed that although anandamide treatment had no observable effect 2-arachidonylglycerol treatment effectively dissipated the lipid raft structures and caused the lipid raft-associated proteins lyn and flotillin-1 to disperse into the surrounding membrane. In addition, anandamide, but not 2-arachidonylglycerol, induced an accumulation of ceramide, which was required for anandamide-induced suppression of cell growth. Finally we demonstrated that anandamide and ceramide treatment of cholangiocarcinoma cells recruited Fas and Fas ligand into the lipid rafts, subsequently activating death receptor pathways. These findings suggest that modulation of the endocannabinoid system may be a target for the development of possible therapeutic strategies for the treatment of this devastating cancer.

The role of the thyroid hormone agonist 3,3',5 l-tri-iodothyronine (T3) on cholangiocytes is unknown. We evaluated the in vivo and in vitro effects of T3 on cholangiocyte proliferation of bile duct-ligated (BDL) rats. We assessed the expression of alpha(1)-, alpha(2)-, beta(1)-, and beta(2)-thyroid hormone receptors (THRs) by immunohistochemistry in liver sections from normal and BDL rats. BDL rats were treated with T3 (38.4 mug/day) or vehicle for 1 wk. We evaluated 1) biliary mass and apoptosis in liver sections and 2) proliferation in cholangiocytes. Serum-free T3 levels were measured by chemiluminescence. Purified BDL cholangiocytes were treated with 0.2% BSA or T3 (1 muM) in the absence/presence of U-73122 (PLC inhibitor) or BAPTA/AM (intracellular Ca(2+) chelator) before measurement of PCNA protein expression by immunoblots. The in vitro effects of T3 (1 muM) on 1) cAMP, IP(3), and Ca(2+) levels and 2) the phosphorylation of Src Tyr139 and Tyr530 (that, together, regulate Src activity) and ERK1/2 of BDL cholangiocytes were also evaluated. alpha(1)-, alpha(2)-, beta(1)-, and beta(2)-THRs were expressed by bile ducts of normal and BDL rats. In vivo, T3 decreased cholangiocyte proliferation of BDL rats. In vitro, T3 inhibition of PCNA protein expression was blocked by U-73122 and BAPTA/AM. Furthermore, T3 1) increased IP(3) and Ca(2+) levels and 2) decreased Src and ERK1/2 phosphorylation of BDL cholangiocytes. T3 inhibits cholangiocyte proliferation of BDL rats by PLC/IP(3)/Ca(2+)-dependent decreased phosphorylation of Src/ERK1/2. Activation of the intracellular signals triggered by T3 may modulate the excess of cholangiocyte proliferation in liver diseases.

Objectives: The aim of the study was to describe the effect of motor cortex stimulation (MCS) on pain thresholds in deafferentated rats., Settings and Design: The effect of MCS was studied in 18 deafferentated and 14 intact laboratory rats, using a standardised plantar test and tail-flick latencies. Two inoxious stimulation electrodes were implanted subdurally over the cerebral cortex and a C5-Th1 dorsal root rhizotomy was performed on the left side. Pain thresholds were measured before and after cortical stimulation. The data were analysed with ANOVA for repeated measures., Results: MCS in intact animals evoked no changes in pain thresholds except for the contralateral forelimb, in which the pain threshold increased after MCS. Following deafferentation, pain thresholds increased in both plantar test and tail-flick in comparison to baseline values. When MCS was applied to the deafferentated animals, the pain thresholds returned to baseline levels. The effect of MCS disappeared within 24 hours., Main Findings: 1. MCS in intact animals evoked hypoesthesia in the corresponding contralateral forelimb; 2. deafferentation itself increased pain thresholds in the unaffected limbs; 3. under MCS, pain thresholds in deafferentated rats were not different from pre-dafferentation values; 4. the effect of MCS disappeared in 24 hours and oscillated., Conclusions: Our results show a similar effect of the stimulation in man and experimental animals despite the differences in the organisation of the cerebral cortex. The use of laboratory animals is promising for further studies in the field of involved antalgic mechanisms of MCS.

Objectives: The aim of the study was to describe the development of self-mutilation after extensive dorsal rhizotomy of the brachial plexus performed during early ontogeny in rats., Settings and Design: The rhizotomy was performed in three groups of rats according to the central nervous system maturation: infant, young, and adult. After the surgery the occurrence of self-mutilation behavior was compared. Rats from the infant group and non-mutilating deafferentated rats from the adult group underwent extracellular recordings from intralaminar thalamic neurons. Interspikes intervals of the records were compared by means of chaodynamic methods., Results: In the infant group self-mutilation did not develop at all. Among the young group self-mutilation developed in 40% of rats and consisted of superficial wounds in all cases. In adult self-mutilation appeared in 80% rats and consisted of both superficial wounds (75%) and amputation (25%). In the newborn group and the deafferentated adult group without any signs of self-mutilation means of the parameters were not significantly different and were significantly lower than those of intact adult rats., Main Findings: 1. Self-mutilation does not develop after the rhizotomy in the infant rats. 2. Neurons behave in chaotic way in adult as well as in young animals. 3. Chaodynamic parameters do not differ between infant and adult rats without any signs of self-mutilation., Conclusions: The results suggest that development of self-mutilation behavior in rats strongly depends on the ontogenetical period of nervous system injury, and that mature nervous system is required for the development of described pathological behavior.

Objectives: A novel evaluative approach was used to determine single unit activities of non-bursting intralaminar thalamic nuclei under neuropathic pain state following dorsal rhizotomy., Settings and Design: Extensive dorsal rhizotomy at cervicothoracic level in rats was used as a model of central pain. After rhizotomy, rats were divided into two groups: rats without any signs of self-mutilation, and those presenting self-mutilation. Spontaneous single unit activities of neurons of intralaminar thalamic nuclei were recorded and interspike intervals (ISIs) of non-bursting cells were counted for both groups and compared with that of non-rhizotomized control rats. Chaodynamic methods were applied for the evaluation of the ISIs., Results: In control rats Lyapunov exponents, Shannon entropy and mutual information average values were significantly higher than those of rhizotomized rats without any signs of self-mutilation. Paradoxically, in animals presenting self-mutilation following rhizotomy the evaluated parameters were similar to those of controls. Further, Lyapunov exponents were positive values in all animals indicating chaotic pattern of the neuronal firing., Main Findings: 1. Neurons behave in chaotic way in all animals, 2. The most regular firing was found in non-mutilating rhizotomized animals, 3. Patterns of the firing in selfmutilating rats were similar to those in controls., Conclusions: It is concluded that pain feeling is not executed neither by changes of chaotic dynamics of non-bursting intralaminar thalamic neurons. On the other hand, the paradoxical firing of the neurons under pathological brain matrix might participate in modification pain feeling.