Intradiscal delivery of celecoxib-loaded microspheres restores intervertebral disc integrity in a preclinical canine model.

Low back pain, related to degeneration of the intervertebral disc (IVD), affects millions of people worldwide. Clinical studies using oral cyclooxygenase-2 (COX-2) inhibitors have shown beneficial effects, although side-effects were reported. Therefore, intradiscal delivery of nonsteroidal anti-inflammatory drugs can be an alternative treatment strategy to halt degeneration and address IVD-related pain. In the present study, the controlled release and biologic potency of celecoxib, a selective COX-2 inhibitor, from polyesteramide microspheres was investigated in vitro. In addition, safety and efficacy of injection of celecoxib-loaded microspheres were evaluated in vivo in a canine IVD degeneration model. In vitro, a sustained release of celecoxib was noted for over 28 days resulting in sustained inhibition of inflammation, as indicated by decreased prostaglandin E2 (PGE2) production, and anti-catabolic effects in nucleus pulposus (NP) cells from degenerated IVDs on qPCR. In vivo, there was no evidence of adverse effects on computed tomography and magnetic resonance imaging or macroscopic evaluation of IVDs. Local and sustained delivery of celecoxib prevented progression of IVD degeneration corroborated by MRI, histology, and measurement of NP proteoglycan content. Furthermore, it seemed to harness inflammation as indicated by decreased PGE2 tissue levels and decreased neuronal growth factor immunopositivity, providing indirect evidence that local delivery of a COX-2 inhibitor could also address pain related to IVD degeneration. In conclusion, intradiscal controlled release of celecoxib from polyesteramide microspheres prevented progression of IVD degeneration both in vitro and in vivo. Follow-up studies are warranted to determine the clinical efficacy of celecoxib-loaded PEAMs in chronic back pain.

Controlled release of celecoxib inhibits inflammation, bone cysts and osteophyte formation in a preclinical model of osteoarthritis.

Major hallmarks of osteoarthritis (OA) are cartilage degeneration, inflammation and osteophyte formation. COX-2 inhibitors counteract inflammation-related pain, but their prolonged oral use entails the risk for side effects. Local and prolonged administration in biocompatible and degradable drug delivery biomaterials could offer an efficient and safe treatment for the long-term management of OA symptoms. Therefore, we evaluated the disease-modifying effects and the optimal dose of polyesteramide microspheres delivering the COX-2 inhibitor celecoxib in a rat OA model. Four weeks after OA induction by anterior cruciate ligament transection and partial medial meniscectomy, 8-week-old female rats (n = 6/group) were injected intra-articular with celecoxib-loaded microspheres at three dosages (0.03, 0.23 or 0.39 mg). Unloaded microspheres served as control. During the 16-week follow-up, static weight bearing and plasma celecoxib concentrations were monitored. Post-mortem, micro-computed tomography and knee joint histology determined progression of synovitis, osteophyte formation, subchondral bone changes, and cartilage integrity. Systemic celecoxib levels were below the detection limit 6 days upon delivery. Systemic and local adverse effects were absent. Local delivery of celecoxib reduced the formation of osteophytes, subchondral sclerosis, bone cysts and calcified loose bodies, and reduced synovial inflammation, while cartilage histology was unaffected. Even though the effects on pain could not be evualated directly in the current model, our results suggest the application of celecoxib-loaded microspheres holds promise as novel, safe and effective treatment for inflammation and pain in OA.

Surface-modified nanoparticles as a new, versatile, and mechanically robust nonadhesive coating: suppression of protein adsorption and bacterial adhesion.

The synthesis of surface-modified silica nanoparticles, chemically grafted with acrylate and poly(ethylene glycol) (PEG) groups, and the ability of the resulting crosslinked coatings to inhibit protein adsorption and bacterial adhesion are explored. Water contact angles, nanoindentation, and atomic force microscopy were used to characterize the cross-linked coatings. Coatings showed a high degree of hydrophilicity combined with a remarkable hardness and stiffness in the dry state. Adsorption of the small protein lysozyme from buffer solution on coated silica wafers decreased significantly with increasing grafting density of the PEG groups on the nanoparticles and was completely inhibited at 0.6 chains nm(-2). Coatings significantly reduced adhesion of Staphylococcus epidermidis HBH 276 in a parallel plate flow chamber with respect to bare glass (>90%), whereas adhesion of Pseudomonas aeruginosa AK1 was only marginally affected by the presence of the coating (<15%). Passage of an air-bubble resulted in almost complete detachment (>93%) of both strains from coated glass, indicating that the adhesion strength between both bacterial strains and the coated surface was significantly reduced by the grafted PEG groups. These coatings thus provide a new method to prepare mechanically robust films with nonadhesive properties that will be extremely useful for the design of biocompatible surfaces in biomedical applications.

[Arterial back table pressure perfusion prevents ischemic biliary lesions after orthotopic liver transplantation]. / Arterielle Ex-situ-Nachperfusion unter Druck zur Vermeidung von "Ischemic Type Biliary Lesions" nach orthotoper Lebertransplantation.

INTRODUCTION: Ischemic biliary lesions are a threatening complication following orthotopic liver transplantation. Their exact pathophysiological mechanism is unknown so far, but insufficient perfusion of biliary arterial vessels might be responsible for the development of these lesions. This might be changed by improved perfusion techniques. We performed a controlled study of cases since February 2000. MATERIALS AND METHODS: We used arterial back table pressure perfusion to achieve reliable perfusion of the capillary system of the biliary tract, which may be impaired by the high viscosity of University of Wisconsin solution. In this study, 190 orthotopic liver transplantations performed between September 1997 and July 2002 were investigated with regard to ischemic biliary lesions. RESULTS: One hundred thirty-one grafts were preserved by in situ standard perfusion including portal perfusion,whereas additional arterial back table pressure perfusion was performed in 59 cases. Donor-related factors, recipient age, indications for transplantation, transplantation techniques, and ischemia times were comparable between groups. Twenty-one (16%) of the patients in the standard perfusion group and only one of the those receiving arterial back table pressure perfusion developed ischemic biliary lesions. This difference was highly significant (P=0.004). Maximal aspartate aminotransferase and alanine aminotransferase levels in the first 3 days were significantly lower in the arterial back table pressure perfusion group (P>0.05). CONCLUSION: Arterial back table pressure perfusion is an easy and reliable method for preventing ischemic biliary lesions in orthotopic liver transplantation. It should, therefore, be the standard technique in liver procurement.

Influence of N-acetylcysteine on hepatic amino acid metabolism in patients undergoing orthotopic liver transplantation.

Experimental treatment with the antioxidant and glutathione precursor N-acetylcysteine (NAC) has been performed in orthotopic liver transplantation (OLT) to reduce reperfusion injury. To investigate the effect of NAC on the hepatic and intestinal amino acid metabolism, intraoperative amino acid exchange rates were studied in liver transplant recipients with high dose NAC treatment (n = 10) and in control patients (n = 9). Treatment with NAC was found to cause a loss of amino acids and increased urea nitrogen release from the liver graft. The net balance of most amino acids was shifted to increased hepatic release or decreased hepatic uptake. The initial cumulative splanchnic release of all proteinogenic amino acids in the NAC treated group was significantly higher than in the control group. These findings are tentatively explained by an increased net protein catabolism in the liver. The increased hepatic urea and glutamine production rate of the NAC treated patients is expected to increase the energy and oxygen demand of the liver in this critical situation. Thus, NAC may have caused marked metabolic disturbances in the freshly implanted graft. The dosage of NAC should therefore be modified to avoid these disadvantages.

N-acetylcysteine attenuates the increase in alpha-glutathione S-transferase and circulating ICAM-1 and VCAM-1 after reperfusion in humans undergoing liver transplantation.

BACKGROUND: Oxidative stress and leukocyte-endothelial interactions contribute significantly to the reperfusion injury of the transplanted liver. Therefore, we investigated the effect of N-acetylcysteine (NAC) on reperfusion injury and circulating adhesion molecules during human liver transplantation. METHODS: In a prospective study, 10 orthotopic liver transplantation patients were treated with high-dose NAC and 10 patients were treated with 5% glucose (placebo group) immediately before and during reperfusion of the donor liver. Parameters of hepatocellular injury, cellular oxygenation, plasma cytokines, and circulating adhesion molecules were determined at various time points during the liver transplantation. RESULTS: NAC had no significant effect on the arterial lactate/pyruvate or hydroxybutyrate/acetoacetate ratio during the liver transplantation. At baseline, liver transplantation patients exhibited elevated levels of cytokines and circulating adhesion molecules compared with healthy volunteers (n=7). While no significant effect of NAC on circulating L- and P-selectin was observed, it significantly inhibited the increase in circulating ICAM-1 and VCAM-1 24 hr after reperfusion. There were no significant differences in maximal postoperative values of serum aspartate transaminase (peak AST) or alanine transaminase (peak ALT) between both groups. However, NAC significantly reduced the rise in alpha-glutathione S-transferase after reperfusion of the donor liver. CONCLUSIONS: NAC attenuated the increase in alpha-glutathione S-transferase and circulating ICAM-1 and VCAM-1 after reperfusion of the donor liver, indicating possible cytoprotective effects of NAC.

N-acetylcysteine induces shedding of selectins from liver and intestine during orthotopic liver transplantation.

In orthotopic liver transplantation (OLT), N-acetylcysteine (NAC) reduces ischaemia/reperfusion (I/R) injury, improves liver synthesis function and prevents primary nonfunction of the graft. To further elucidate the mechanisms of these beneficial effects of NAC, we investigated influence of high-dose NAC therapy on the pattern of adhesion molecule release from liver and intestine during OLT. Nine patients receiving allograft OLT were treated with 150 mg NAC/kg during the first hour after reperfusion; 10 patients received the carrier only. One hour after reperfusion, samples of arterial, portal venous and hepatic venous plasma were taken and blood flow in the hepatic artery and the portal vein was measured. Absolute concentrations of sICAM-1, sVCAM-1, sP-selectin and sE-selectin were not markedly different. However, balance calculations showed release of selectins from NAC-treated livers as opposed to net uptake in controls (P < or = 0.02 for sP-selectin). This shedding of selectins might be a contributing factor to the decrease in leucocyte adherence and improved haemodynamics found experimentally with NAC-treatment.

The efficacy of N-acetylcysteine as a hepatoprotective agent in liver transplantation.

One of the most common complications after liver transplantation is primary graft dysfunction which results from severe deterioration of the microcirculation. The data obtained from our experimental studies indicate that N-acetylcysteine (NAC) is able to reduce the severity of ischemia/reperfusion injury and improves postoperative graft function after liver transplantation in rats. The aim of this pilot study was to evaluate the efficacy of NAC as a hepatoprotective agent under clinical conditions. A group of 30 liver transplanted patients were treated with NAC, and 30 patients (control group) were treated with a 5% solution of glucose only. In the NAC group we observed a distinct reduction in ischemia/reperfusion injury and improved liver function with less elevated peak transaminases, better macrocirculation, improved liver synthesis function and a lower incidence of primary nonfunction compared with the control group. We conclude that NAC is a very promising substance for reducing graft dysfunction in clinical liver transplantation.

Extrahepatic biliary obstruction impairs microvascular perfusion and increases leukocyte adhesion in rat liver.

To determine if disturbances of the liver microcirculation may be of pathophysiological relevance for liver damage during acute biliary obstruction, we studied the effects of bile duct ligation (BDL) on hepatic microhemodynamics and leukocyte adhesion in rat liver in vivo. Male Wistar rats were subjected to BDL for 3 days and 7 days, respectively. Sham-operated controls underwent laparotomy without BDL. After 3 days, intravital fluorescence microscopy (IVM) and hydrogen gas (H2) clearance were performed to study hepatic microvascular perfusion. Furthermore, leukocyte-endothelial cell interactions were assessed by IVM. Intercellular adhesion molecule 1 (ICAM-1) protein expression was studied by Western blot analysis and tissue immunofluorescence after 3 and 7 days, respectively. Analysis of microvascular perfusion by IVM revealed a marked impairment of sinusoidal perfusion after 3 days. Assessment of H2 clearance confirmed that overall hepatic microvascular perfusion was decreased. In addition, increased leukocyte adhesion in sinusoids and venules could be observed. A concomitant increase of ICAM-1 expression in liver tissue was also noted within the first week after BDL. Our results show that BDL is followed by a marked depression of the hepatic microcirculation and increased leukocyte adhesion in vivo within 3 to 7 days. Together, these findings suggest that deficits in microvascular perfusion and increased neutrophil infiltration may represent a potential source of liver injury during acute biliary obstruction.

Inhibition of nitric oxide synthesis in ischemia/reperfusion of the rat liver is followed by impairment of hepatic microvascular blood flow.

BACKGROUND: Recent studies provide evidence that nitric oxide (NO) has beneficial effects in hepatic ischemia/reperfusion injury. The purpose of this study was to evaluate whether nitric oxide is involved in the regulation of hepatic microvascular perfusion after warm hepatic ischemia. Therefore, we performed a study using in vivo fluorescence microscopy. METHODS: Clamping of the left liver lobe was performed in male Wistar rats for the duration of 70 min. One experimental group (n=8) received L-NAME (Nw-nitro-L-arginine methyl ester hydrochloride), an NO-synthase inhibitor, 1 min prior to reperfusion. A second experimental group (n=8) received L-arginine (NO-substrate) continuously infused throughout the observation period. Controls (n=8) received equivalent volumes of an isotonic solution and underwent the same procedures. Hepatic microvascular blood flow and leukocyte-endothelial cell interaction was studied between 20 and 90 min after reperfusion using in vivo fluorescence microscopy. RESULTS: Inhibition of NO-synthesis during reperfusion by application of L-NAME caused a marked decrease in sinusoidal blood flow velocity. Furthermore, we noted an increase of non-perfused sinusoids in this group. Treatment with L-arginine improved functional perfusion of hepatic acini and reduced significantly the number of adherent leukocytes in sinusoids and venules compared to control animals. CONCLUSIONS: Our results provide further evidence that NO maintains postischemic hepatic microvascular perfusion and that inhibition of NO synthesis has detrimental effects on hepatic microhemodynamics during reperfusion.

Effects of mixed ETA and ETB-receptor antagonist (Ro-47-0203) on hepatic microcirculation after warm ischemia.

There is evidence that endothelin (ET) is involved in disturbances of the hepatic microcirculation after warm ischemia. In this study we investigated the influence of a mixed ETA-, ETB-receptor antagonist (Bosentan) on ischemia-reperfusion damage of the liver by means of intravital fluorescence microscopy (IVM). Clamping of the left liver lobe (= warm ischemia) was performed in 16 male Wistar rats for 70 min. The treatment group (N = 8) received 15 mg/kg Bosentan (Ro-47-0203) 1 min prior to reperfusion. Controls (N = 8) received an equivalent amount of Ringer's solution. Between 20 and 90 min after reperfusion, leukocyte-endothelial cell interactions in sinusoids and postsinusoidal venules as well as perfusion of hepatic acini were studied. Application of Bosentan improved sinusoidal blood flow, attenuated manifestations of microvascular perfusion failure, and decreased the number of rolling leukocytes in postsinusoidal venules. Our results provide further evidence that ET is involved in postischemic impairment of hepatic microhemodynamics during reperfusion.

Improvement of hepatic microhemodynamics by N-acetylcysteine after warm ischemia.

In this study we investigated the influence of N-acetylcysteine (NAC) on the hepatic microcirculation after warm ischemia by intravital fluorescence microscopy. Clamping of the left liver lobe was performed in 20 male Wistar rats for 70 min. The treatment group (n = 10) received 400 mg NAC/kg body weight 20 min prior to clamping. After reperfusion, acinar and sinusoidal perfusions were observed as well as the leukocyte-endothelium interaction. Phagocytic activity was assessed after application of latex beads. NAC reduced the number of nonperfused sinusoids in all acinar zones. A reduction in zone 1 (portal) was achieved from 15.5 to 7.1% (p < 0.0001), in zone 2 (midzonal) from 14.6 to 6.1% (p < 0.0001) and in zone 3 (central) from 11.9 to 2.9% (p < 0.0001). There were no significant differences in leukocyte adherence as well as in phagocytic activity detectable. We conclude that NAC improves hepatic microcirculation after warm ischemia by increasing sinusoidal blood flow.

Impact of N-acetylcysteine on the hepatic microcirculation after orthotopic liver transplantation.

Recent observations showed an improvement of hepatic macro- and microhemodynamics as well as survival rates after warm ischemia of the liver following treatment with N-acetylcysteine (NAC). In this study we assessed the influence of NAC on the hepatic microcirculation after orthotopic liver transplantation (OLT) using intravital fluorescence microscopy. OLT with simultaneous arterialization was performed in 16 male Lewis rats following cold storage in University of Wisconsin solution for 24 hr. Within the experimental group (n = 8) donors received NAC (400 mg/kg) 25 min before hepatectomy. In addition, high-dose treatment of recipients with NAC (400 mg/kg) was started with reperfusion. Control animals (n = 8) received an equivalent amount of Ringer's solution. Intravital fluorescence microscopy was performed 30-90 min after reperfusion assessing acinar and sinusoidal perfusion, leukocyte-endothelium interaction, and phagocytic activity. Treatment with NAC reduced the number of nonperfused sinusoid from 52.4 +/- 0.8% to 15.7 +/- 0.5% (p = 0.0001) (mean +/- SEM). Furthermore, we achieved a significant reduction of leukocytes adhering to sinusoidal endothelium (per mm2 liver surface) from 351.9 +/- 13.0 in controls to 83.6 +/- 4.2 in the experimental group (P = 0.0001). In postsinusoidal venules, treatment with NAC decreased the number of sticking leukocytes (per mm2 endothelium) from 1098.5 +/- 59.6 to 425.9 +/- 37.7 (P = 0.0001). Moreover, bile flow was significantly increased after therapy with NAC (4.3 +/- 1.2 vs. 2.2 +/- 0.7 ml/90 min x 100g liver) (P < 0.05). Phagocytic activity was not influenced by application of NAC. We conclude that high-dose therapy with NAC in OLT attenuates manifestations of microvascular perfusion failure early after reperfusion and should be considered as a means to reduce reperfusion injury.

Prolongation of graft survival in allogeneic pancreas and liver transplantation by (-)15-deoxyspergualin.

(-)15-Deoxyspergualin, originally discovered as an antitumoral drug, was shown to have different immunosuppressive effects, when pancreas and orthotopic allogeneic liver transplantations in rats were compared. In the strong rejection model dark agouti----Lewis (RT1a----RT1(1)) we could only show a minor immunosuppressive effect, as far as pancreaticoduodenal and pancreas segment transplantations are concerned: graft survival was prolonged by 9 days in pancreas segment allografts (p less than 0.01) and by 6 days in pancreaticoduodenal allografts (p less than 0.01), when recipients were treated by ten doses of 2.5 mg/kg deoxyspergualin. Pretreatment of recipients with 15-deoxyspergualin was not efficient. On the contrary, in orthotopic liver transplantation done by the cuff technique, a remarkable prolongation of allograft survival could be demonstrated: about half of the animals showed prolongation of allograft survival for more than 80 days, compared with about 11 days in the control group (p less than 0.01). The substance is considered to be valuable for clinical application.