Compression of Vectors for Small Interfering RNAs Delivery: Toward Oral Administration of siRNA Lipoplexes in Tablet Forms.

Currently, most nonviral nucleic acid vectors are in the form of colloidal suspensions administered primarily parenterally. This type of formulation and the mode of administration impose strong constraints such as the size of the administered vectors or the production of sterile preparations. The tablet form provides access to easy oral administration, well accepted by patients; As regards nucleic acid vectors, a dry form represents an advance in terms of stability. Using an optimized lipid-based small interfering RNA-delivery system, we studied the tabletability of a liquid suspension of these vectors. We optimized the conditions of freeze-drying by choosing excipients and process, allowing for the conservation of both the gene-silencing efficacy of the formulated siRNAs and the supramolecular structure of the lipid particulate system. Gene-silencing efficacy was assayed on luciferase-expressing cells and the structure of the siRNA vector in freeze-dried and tablet forms was examined using small-angle X-ray scattering (SAXS) synchrotron radiation. The freeze-dried powders were then mixed with excipients necessary for the good progress of the compression by allowing for a regular supply of the matrix and the reduction of friction. The compression was carried out using a rotary press simulator that allows for complete monitoring of the compression conditions. After compression, formulated siRNAs retained more than 60% of their gene-silencing efficacy. Within the tablets, a specific SAXS signal was detectable and the lamellar and cubic phases of the initial liquid suspension were restored after resuspension of siRNA vectors by disintegration of the tablets. These results show that the bilayer lipid structures of the particles were preserved despite the mechanical constraints imposed by the compression. If such a result could be expected after the freeze-drying step, it was never shown, to our knowledge, that siRNA-delivery systems could retain their efficacy and structure after mechanical stress such as compression. This opens promising perspectives to oral administration of siRNA as an alternative to parenteral administration.

Alternative routes for parenteral nucleic acid delivery and related hurdles: highlights in RNA delivery.

INTRODUCTION: Nucleic acid-based therapies are promising advancements in medicine. They offer unparalleled efficacy in treating previously untreatable diseases through precise gene manipulation techniques. However, the challenge of achieving targeted delivery to specific cells remains a significant obstacle. AREAS COVERED: This review thoroughly examines the physicochemical properties of nucleic acids, focusing on their interaction with carriers and exploring various delivery routes, including oral, pulmonary, ocular, and dermal routes. It also examines the nonviral vector delivery efficiency of nucleic acids, focusing on RNA, and provides regulatory landscapes. EXPERT OPINION: The role of carriers in improving the effectiveness of nucleic acid-based therapies is emphasized. The discussion of published results covers regulatory frameworks, including insights into European Medicines Agency guidelines. It highlights cutting-edge biotechnological innovations and a quality-by-design approach that could facilitate clinical translation and smooth regulatory obstacles.

Best temperature for static liver graft storage is 1°C.

BACKGROUND: The best storage temperature in liver transplantation remains an unsolved question. METHODS: After storage for 24h in University of Wisconsin solution at +4°C, +1°C, or -0.5°C, rat livers were subjected, or not, to 15min of warm ischemia, rinsed with Ringer lactate, and subsequently reperfused with oxygenated Krebs-Henseleit buffer. RESULTS: In the presence of warm ischemia, for livers stored at +4°C, creatine kinase (CK) peaked at 21±5IUg(-1)h(-1), hepatic resistance at 34,700±1500dynscm(-5), bile flow reached 18±4µLg(-1)h(-1) after 10min, and oxygen consumption stabilized at about 25µmolg(-1)h(-1) after 20min. When livers were stored at +1°C, CK and hepatic resistance were lowered, bile production was 33±6µLg(-1)h(-1) (P<0.05 versus +4°C), and oxygen consumption was 105±10µmolg(-1)h(-1) (P<0.001). For livers stored at-0.5°C, results were not statistically different from those of the +1°C group except for bile flow, which was significantly lower. Without warm ischemia, the peak of CK (P=0.015) and the peak hepatic resistance (P<0.001) of the +4°C group were significantly increased compared with the +1°C or -0.5°C groups. However, no difference in bile flow or oxygen consumption was observed. The number of trypan blue-positive nonparenchymal cells (P=0.003) and the gain in liver weight during the reperfusion (P=0.015) were minimal after storage at +1°C. CONCLUSIONS: Static storage at +1°C improved liver function compared with +4°C or -0.5°C. Main beneficial effect was observed with parameters reflecting sinusoidal cells injury.

[Topical treatment for age-related macular degeneration: Where are we now?] / Traitement topique de la dégénérescence maculaire liée à l'âge - Où en sommes-nous ?

The therapeutic management of age-related macular degeneration (AMD) is a major public health issue. One of its two late forms, neovascular AMD, is currently treated by intravitreal injections of pharmaceutical active ingredients. Although it is very effective in treating pathologies of the posterior segment of the eye, the intravitreal route is not an ideal option for the long-term management of a chronic disease such as AMD. Indeed, in the literature, some authors even call it a "burden" for the practitioners, the patients and the healthcare system. Thus, consideration should be given to less invasive routes. Among the possible administration routes to reach the posterior segment of the eye, the most suitable for the patient with the least risk of systemic adverse effects is the topical route. Several research teams have attempted to formulate molecules for topical administration in the treatment of atrophic or neovascular AMD. In this review, we emphasize the importance of the pharmaceutical formulation to meet the challenge of targeting the posterior segment of the eye by a topical route.

Title: Traitement topique de la dégénérescence maculaire liée à l'âge - Où en sommes-nous ? Abstract: La prise en charge thérapeutique de la dégénérescence maculaire liée à l'âge (DMLA) est un enjeu majeur de santé publique. L'une de ses deux formes tardives, la DMLA néovasculaire, est actuellement traitée par injection intravitréenne de molécules anti-angiogéniques. Bien qu'elle soit très efficace pour traiter les atteintes du segment postérieur de l'œil, la voie intravitréenne n'est pas une option idéale pour la prise en charge au long cours d'une maladie chronique telle que la DMLA. L'administration topique de molécules actives contre cette maladie, plus confortable pour le patient et moins coûteuse pour la société, représente un vrai défi.

Arginine reduces bacterial invasion in rats with head injury: an in vivo evaluation by bioluminescence.

OBJECTIVES: The benefit of arginine in intensive care unit patients with severe sepsis is still controversial. An excessive supply of arginine could lead to an overproduction of nitric oxide and could be responsible for septic shock and multiorgan failure. However, this claim is not supported by any experimental or clinical data. We set out to determine whether an enteral supply of arginine would modulate bacterial invasion in rats with head injury. METHODS: Male Sprague-Dawley rats with head injury were randomized into two groups. Group 1 included rats with head injury fed a standard enteral nutrition (Sondalis HP, n = 10) and group 2 included rats with head injury fed the standard enteral nutrition plus arginine (4 g/kg/d, n = 11). Two days after head injury, the rats received a single enteral bolus of luminescent Escherichia coli Xen 14. Bacterial proliferation was evaluated in vivo at time + 2 hrs and time + 6 hrs after E. coli challenge. Four days after head injury, blood was sampled for arginine and fibrinogen assay. Muscles, intestine, spleen, and thymus were removed and weighed. RESULTS: There was no mortality in either group. The luminescence signal was similar in the two groups at time +2 hrs (group 1: 414 [5-823] vs. group 2: 496 [0.1-993] (median value[min-max]; not significant) and was significantly lower at time +6 hrs in group 2 (group 1: 71 [0-142] vs. group 2: 8.5 [0-17]; p = .026). Arginine treatment did not improve any nutritional parameters. CONCLUSIONS: Arginine was not responsible for mortality in rats with head injury with infectious complications and reduced the intensity of bacterial invasion.

Fate of Tableted Freeze-Dried siRNA Lipoplexes in Gastrointestinal Environment.

The incorporation of siRNA into nanocarriers is mandatory to facilitate its intracellular delivery, as siRNA itself cannot enter cells. However, the incorporation of these nanocarriers into oral, solid dosage forms and their fate in the gastrointestinal environment is yet to be explored. In the present work, the fate of, (i) naked siRNA, (ii) freshly prepared siRNA lipoplexes, and (iii) tableted siRNA lipoplexes, in simulated gastric and intestinal fluids was studied. The siRNA, either released from or protected within the lipoplexes, was quantified by gel electrophoresis and siRNA efficacy was assessed in cell transfection. The freshly prepared lipoplexes kept their siRNA load and transfection efficiency totally preserved during 1 h of incubation in simulated gastric fluid at 37 °C. However, in simulated intestinal fluid, despite no release of siRNA from lipoplexes after 6 h of incubation, gene silencing efficacy was dramatically decreased even after 1 h of exposure. The lipoplexes obtained from tablets efficiently protected siRNA in simulated gastric fluid, thus preserving the gene silencing efficacy, whereas their incubation in simulated intestinal fluid resulted in a marked siRNA release and decreased gene silencing efficacy. These results provided a detailed explanation for understanding the fate of siRNA in gastrointestinal conditions, when simply loaded in lipoplexes or formulated in the form of tablets.

Neuroprotective Effect of siRNA Entrapped in Hyaluronic Acid-Coated Lipoplexes by Intravitreal Administration.

Since the possibility of silencing specific genes linked to retinal degeneration has become a reality with the use of small interfering RNAs (siRNAs), this technology has been widely studied to promote the treatment of several ocular diseases. Despite recent advances, the clinical success of gene silencing in the retina is significantly reduced by inherent anatomical and physiological ocular barriers, and new strategies are required to achieve intraocular therapeutic effectiveness. In this study, we developed lipoplexes, prepared with sodium alginate as an adjuvant and strategically coated with hyaluronic acid (HA-LIP), and investigated the potential neuroprotective effect of these systems in a retinal light damage model. Successful functionalization of the lipoplexes with hyaluronic acid was indicated in the dynamic light scattering and transmission electron microscopy results. Moreover, these HA-LIP nanoparticles were able to protect and deliver siRNA molecules targeting caspase-3 into the retina. After retinal degeneration induced by high light exposure, in vitro and in vivo quantitative reverse transcription-PCR (RT-qPCR) assays demonstrated significant inhibition of caspase-3 expression by HA-LIP. Furthermore, these systems were shown to be safe, as no evidence of retinal toxicity was observed by electroretinography, clinical evaluation or histology.

Freeze-dried fecal samples are biologically active after long-lasting storage and suited to fecal microbiota transplantation in a preclinical murine model of Clostridioides difficile infection.

Fecal microbiota transplantation is now recommended for treating recurrent forms of Clostridioides difficile infection. Recent studies have reported protocols using capsules of either frozen or freeze-dried stool allowing oral administration in in- and out-patient settings. However, a central question remains the viability, engraftment, and efficacy of the microbiome over time during storage life. This study shows that both the freeze-drying and freezing procedures for fecal samples allowed preserving viability, short-chain fatty acids concentration, and anti-Clostridioides difficile properties of microbiota without significant alteration after storage for 12 months. Fecal transplantation with freeze-dried microbiota allowed engraftment of microbiota leading to clearance of Clostridioides difficile infection in a preclinical murine model with a survival rate of 70% versus 53-60% in mice treated with frozen inocula, and 20% in the untreated group. Moreover, the freeze-dried powder can be used to fill oral hard capsules using a very low amount (0.5%) of glidant excipient, allowing oral formulation. Altogether, this study showed that freeze-dried inocula can be used for the treatment of Clostridioides difficile infection with long-lasting stability of the fecal microbiota. This formulation facilitates biobanking and allows the use of hard capsules, an essential step to simplify patient access to treatment.

Hepatic stellate cell hypertrophy is associated with metabolic liver fibrosis.

Hepatic fibrosis is a major consequence of chronic liver disease such as non-alcoholic steatohepatitis which is undergoing a dramatic evolution given the obesity progression worldwide, and has no treatment to date. Hepatic stellate cells (HSCs) play a key role in the fibrosis process, because in chronic liver damage, they transdifferentiate from a "quiescent" to an "activated" phenotype responsible for most the collagen deposition in liver tissue. Here, using a diet-induced liver fibrosis murine model (choline-deficient amino acid-defined, high fat diet), we characterized a specific population of HSCs organized as clusters presenting simultaneously hypertrophy of retinoid droplets, quiescent and activated HSC markers. We showed that hypertrophied HSCs co-localized with fibrosis areas in space and time. Importantly, we reported the existence of this phenotype and its association with collagen deposition in three other mouse fibrosis models, including CCl4-induced fibrosis model. Moreover, we have also shown its relevance in human liver fibrosis associated with different etiologies (obesity, non-alcoholic steatohepatitis, viral hepatitis C and alcoholism). In particular, we have demonstrated a significant positive correlation between the stage of liver fibrosis and HSC hypertrophy in a cohort of obese patients with hepatic fibrosis. These results lead us to conclude that hypertrophied HSCs are closely associated with hepatic fibrosis in a metabolic disease context and may represent a new marker of metabolic liver disease progression.

Evidence for impairment of hepatic energy homeostasis in head-injured rat.

Traumatic brain injury (TBI) is known to induce a metabolic adaptation characterized by a nitrogen transfer from the periphery to the liver. However, the consequences of TBI on liver energy status are poorly documented. We evaluated the consequences of TBI on liver energy homeostasis in rats. In a first set of experiments, rats were randomized into two groups: a TBI group traumatized by fluid percussion, and an ad libitum fed group (AL) of healthy rats. The rats were sacrificed at 2, 3, or 4 days (D2, D3, and D4, respectively to determine the kinetic of hepatic energy changes). Since TBI leads to a profound anorexia, in a second set of experiments TBI rats received enteral nutrition (TBI-EN group) for 4 days to specifically assess the role of anorexia in the hepatic disturbances. TBI led to a decrease in hepatic glycogen (D2: TBI 3.9 +/- 1.9 vs. AL 18.9 +/- 2.6 mg/g, p < 0.05) and ATP (D2: TBI 540 +/- 57 vs. AL 850 +/- 44 nmol/g, p < 0.05) contents. These effects were not linked to anorexia, since they were observed when rats were fed using continuous enteral nutrition. Interestingly, there was no adaptation of the mitochondrial oxidative capacity to compensate for the increase in energy requirements (cytochrome C oxidase activity: AL, 82 +/- 5; TBI, 82 +/- 4; and TBI-EN, 87 +/- 3 micromol/min/g, NS). These findings demonstrate that TBI is responsible for an impairment of liver energy homeostasis. Moreover, these alterations are related neither to anorexia nor to decreased mitochondrial oxidative capacity.

Influence of pH conditions on the viability of Saccharomyces boulardii yeast.

Saccharomyces boulardii is a probiotic with proven health benefits. However its survival is challenged by gastrointestinal transit, and a ratio between 1 and 3% of living yeast is recovered in the feces after oral administration. The aim of the study was to determine to what extent the yeast was sensitive to gastrointestinal pH conditions. Therefore we explored the survival of different concentrations of S. boulardii in conditions mimicking the stomach pH (pH 1.1 0.1 N HCl) and the intestinal pH (pH 6.8 phosphate buffer) in vitro. The probiotic being commercialized as a freeze-dried powder obtained from an aqueous suspension, both forms were evaluated. In phosphate buffer pH 6.8, the viability remained stable for both forms of S. boulardii for 6 h. In HCl pH 1.1, viability of both forms (200 mg L(-1)) significantly decreased from 5 min. Observation under scanning/transmission electron microscopy showed morphological damages and rupture of the yeast wall. Threshold value from which S. boulardii viability was unaltered was pH 4. At the highest concentration of 200 g L(-1), the initial pH value of 1.1 rose to 3.2, exerting a protective effect. In conclusion, although the yeast in aqueous suspension was less sensitive than the freeze-dried yeast to acidic conditions, a gastric protection for improvement of oral bioavailability of viable S. boulardii appears necessary.

Use of Resveratrol Self-Emulsifying Systems in T/C28a2 Cell Line as Beneficial Effectors in Cellular Uptake and Protection Against Oxidative Stress-Mediated Death.

Osteoarthritis (OA) is the most prevalent rheumatic disease in the world. Although its etiology is still unknown, one of the key processes in OA progression and development is oxidative stress. In this context, resveratrol, a well-known anti-oxidant from the stilbene family, could be of particular interest in future OA therapeutic strategies. However, currently, because of its low bioavailability, use of resveratrol in human health is very limited. In this study, we tested two resveratrol self-emulsifying systems previously developed in our laboratory in order to determine if they could improve cellular uptake of resveratrol in a human immortalized chondrocytic cell line (T/C28a2) and enhance protection against oxidative stress. Our results showed that resveratrol self-emulsifying systems were able first to increase cellular tolerance towards resveratrol, and thus decrease resveratrol intrinsic cellular toxicity, allowing the use of higher concentrations, second, to increase resveratrol uptake in membrane and intracellular fractions, and finally, to improve protection against oxidative stress-mediated death in human immortalized chondrocytic cell line T/C28a2. These data suggest that new formulations of resveratrol could be considered as potential beneficial effectors in future OA treatments.

Head injury profoundly affects gut microbiota homeostasis: Results of a pilot study.

OBJECTIVES: Head injury (HI) induces a hypercatabolic state, dysimmunity, and septic complications that increase morbidity and mortality. Although compromised immune function is usually incriminated in infection occurrence, gut dysbiosis could also be involved in this phenomenon and, to our knowledge, has never been considered. To assess if HI could affect microbiota, we explored the impact of HI on intestinal microbiota in a rodent model of fluid percussion. METHODS: Nineteen rats were randomly assigned to two groups: Healthy rats fed ad libitum (n = 7) and HI rats (n = 12), which received standard enteral nutrition for 4 d. Four days after HI, rats were euthanized and cecal contents were sampled. Cecal microbiota was assessed using real-time quantitative polymerase chain reaction. RESULTS: HI significantly decreased the cecal content of strict anaerobic groups, Bacteroides/Prevotella group (HI 8.9 versus healthy controls 9.3 median log10 colony forming units [CFU]/g, P = 0.007), Clostridium cluster XIVab (HI 7.9 versus healthy controls 8.9 median log10 CFU/g, P = 0.002), Lactobacillus/Leuconostoc group (HI 8.5 versus healthy controls 9.4 median log10 CFU/g, P = 0.044), and Bifidobacterium sp. (HI 3.0 versus healthy controls 8.2 median log10 CFU/g, P < 0.001). In contrast, colonization by Escherichia coli was dramatically increased (HI 10.5 versus healthy controls 7.0 median log10 CFU/g, P < 0.001). CONCLUSIONS: HI profoundly modified the gut microbiota homeostasis and thus could contribute to infection in head trauma patients. These preliminary results open a new field of research in the management of patients with HI.

Effect of an immune-enhancing diet on lymphocyte in head-injured rats: what is the role of arginine?

OBJECTIVE: The benefit of immune-enhancing diets (IEDs) in the intensive care unit remains controversial. Considering their complexity, the role of each component, in particular arginine (Arg), in their properties is largely unknown. The aim of this study was to determine the role of arginine in the immunomodulatory effects of an IED (Crucial) in head-injured rats. DESIGN: Thirty-four rats were randomized into five groups: AL (ad libitum), HI (head-injured), HI-STD (HI + standard enteral nutrition, EN), HI-STD-Arg (HI + standard EN + Arg in equimolar concentration to Arg in IED), and HI-IED (HI + IED). These isocaloric and isonitrogenous diets were administered over 4 days. After death, the thymus was removed and weighed. The density of CD25, CD4 and CD8 on lymphocytes from blood and from Peyer patches was evaluated. Mesenteric lymph nodes, liver and spleen were cultured for analysis of enterobacterial translocation and dissemination. MEASUREMENTS AND RESULTS: HI induced an atrophy of the thymus which was not corrected by the standard diet (HI 0.27 +/- 0.03, HI-STD 0.35 +/- 0.03 vs. AL 0.49 +/- 0.02 g; p < 0.05). However, the standard diet supplemented with arginine limited the thymic atrophy and the IED restored thymus weight. CD25 density and interleukin-2 production were increased only in the HI-STD-Arg and HI-IED groups (p < 0.05). Head injury induced enterobacterial translocation and dissemination which were blunted only in the HI-STD-Arg group (p < 0.05). CONCLUSIONS: In this rat HI model, arginine appears to be safe, contributes to a large extent to the immunomodulatory effects of the IED, and seems to limit enterobacterial translocation and dissemination more efficiently alone than in an IED.

Impairment of lymphocyte function in head-injured rats: effects of standard and immune-enhancing diets for enteral nutrition.

BACKGROUND: The metabolic response to head injury (HI) is characterized by a dysimmunity which may be a risk factor of a septic state. The use of immune enhancing diets (IEDs) could be a promising approach to improve immune functions. The aim of the study was to investigate the consequences of HI on lymphocyte function and to determine the effects of an enteral IED comparatively to a standard enteral nutrition. METHOD: A rat model of HI by fluid percussion was used. Twenty-five male Sprague-Dawley rats were randomized into 4 groups: rats receiving standard chow diet ad libitum (AL), rats sustaining HI and receiving standard chow diet and enteral saline (HI), rats receiving the enteral standard diet Sondalis HP (HIS), and rats receiving the IED Crucial (HIC). The two enteral diets were infused continuously during 4 days after the HI and were isocaloric, isonitrogenous and isovolumic. RESULTS: HI induced a thymus atrophy (HI vs. AL, P<0.05), and an impairment in lymphocyte CD25 receptor density responsiveness to stimulation. The IED blunted thymus atrophy and allowed to preserve the stimulation of blood and Peyer patches lymphocytes (HIC: Stimulated vs. Basal, P<0.05). CONCLUSION: IED seems more adapted for preserving lymphocyte function than standard diet in HI patients.

Arginine-enriched diet limits plasma and muscle glutamine depletion in head-injured rats.

OBJECTIVE: Injury is associated with a depletion in glutamine (GLN) pools, which may contribute to impairment of immune and nutritional statuses. Total parenteral nutrition enriched with arginine (ARG) is able to generate GLN in surgical patients. We hypothesized that this same concept may be applicable to enteral administration and could be extended to muscle GLN reserves. This study investigated the ability of an enteral formula enriched with ARG to restore the GLN pools in an experimental model of head injury. METHODS: Twenty-five male Sprague-Dawley rats were randomized into 4 groups: ad libitum access to food, head injury plus free access to nutrition, head injury plus standard enteral nutrition (Sondalis), and an immune-enhancing diet (IED). The two enteral diets were adjusted to be isocaloric (290 kcal.kg(-1).d(-1)) and isonitrogenous (3.29 g.kg(-1).d(-1)) and were delivered for 4 d (24 h/24 h). After sacrifice, plasma and muscle amino acids were determined. RESULTS: Head injury was associated with a large depletion of muscle and plasma GLN pools that were restored by IED administration but not by the standard diet. Moreover, the IED but not the standard diet improved or normalized ornithine and glutamate pools, suggesting that the modification of GLN pools is related to ARG administration. CONCLUSION: In our model of head injury, our IED, a diet without free GLN, is efficient in restoring the plasma and muscle pools of GLN, probably due to its high ARG content.

Evaluation of a new concept of immune-enhancing diet in a model of head-injured rat with infectious complications: A proof of concept study.

Immune-enhancing diet (IED) utilization in critically ill septic patients is still debated. A new concept of IED has been proposed combining extra glutamine sequentially with either antioxidants or other amino acids, in order to match patient requirements according to their response to injury. We evaluated whether this new IED elicits a more favorable response to stress when compared with two existing IEDs both enriched in arginine but with different levels of anti-oxidants, in a validated rat model combining head injury (HI) and infectious complications. Forty-eight HI rats were randomized into four groups (n = 11-13 per group) to receive, for 4 days, standard enteral nutrition (S), one of the two existing IEDs (IED1, IED2), or the new IED (IED3; providing glutamine and antioxidants for two days and glutamine and specific amino acids for two days). Two days after HI, the rats received an enteral bolus of luminescent Escherichia coli Xen14 to induce infection, and bacterial dissemination was evaluated. Body weight (BW) was recorded daily. Four days after HI, animals were euthanized; blood was sampled; organs were weighed; cumulated nitrogen balance (CNB) and nitrogen efficiency were determined. IED3 was more efficient than IED1 and IED2 in improving BW recovery from D3 (D3 vs. D1, p < 0.05) after HI. It significantly improved CNB and net protein utilization (IED3 vs. S, IED1, IED2, p < 0.05). An IED with sequential administration of anti-oxidants and glutamine may be better suited to meeting nutritional requirements in severe catabolic states.

Long-term cold liver storage induces endothelin-1 release and a time-dependent increase in portal pressure at reperfusion in the rat.

BACKGROUND: Long-term cold storage and reperfusion lead to great liver injury involving major microcirculatory disturbance. This study investigated the effect of storage duration on endothelin-1 production, portal pressure, and microcirculation. METHODS: Rat livers were perfused before and after 15 min to 48 h of cold storage in University of Wisconsin (UW) solution. RESULTS: Portal pressure was unchanged for up to 12 h of storage, and increased by 40% and 70% after 24 and 48 h, respectively. Vascular space was increased by a factor of 1.7 following 48 h of storage. Endothelin-1 was released by livers stored in UW solution for 48 h (3.99 +/- 1.85 pg/100 microl). The nonselective endothelin-1 receptor antagonist TAK-044 partially prevented the increase in portal pressure and prevented the increase in vascular space. CONCLUSIONS: Cold storage induces a time-dependent elevated portal pressure at reperfusion. The involvement of endothelin-1 in this process offers opportunities to improve liver graft quality by using endothelin-1 inhibitors.

Isolated perfused liver model: the rat and guinea pig compared.

OBJECTIVE: Although the rat is the most commonly used species for the study of hepatic metabolism, the physiology of the guinea pig is closer to human physiology. We compared the model of isolated perfused guinea pig liver with the classic model of isolated perfused rat liver, especially with respect to amino acid metabolism. METHODS: After validation of an anesthetic mixture of ketamine, diazepam, and xylazine for the guinea pig, isolated perfused livers were harvested for both species. Three groups of animals were compared for the study of liver metabolic fluxes: 6-wk-old male Sprague-Dawley rats (R; 230 +/- 10 g, n = 5), young male Hartley guinea pigs (YG; 223 +/- 8 g, n = 6) matched to rats by liver weight, and adult male Hartley guinea pigs (AG; 389 +/- 5 g, n = 6) matched to rats by age. Results (mean +/- standard error of the mean) were compared by analysis of variance and Newman-Keuls tests. RESULTS: Both models displayed a satisfactory hepatic viability, but differences were noted, with higher portal flows (R: 3.1 +/- 0.3 versus YG: 4.5 +/- 0.3 and AG: 4.2 +/- 0.3 mL. min(-1). g(-1); P < 0.05, YG and AG versus R) and bile flows (R: 0.34 +/- 0.01 versus YG: 2.38 +/- 0.22 versus AG: 3.17 +/- 0.28 microL. min(-1). g(-1); P < 0.05, YG and AG versus R, and YG versus AG) and higher amino acid fluxes (P < 0.05) leading to greater nitrogen uptake (P < 0.05) in guinea pigs. We performed a second set of experiments to evaluate the influence of anesthesia and portal flow on this last parameter. In these experiments, rats were anesthetized with ketamine, diazepam, and xylazine and guinea pig livers were perfused at rat blood flow. Apart from a 50% anesthesia-related mortality for rats, bile flow and metabolic parameters were only slightly modified. However, some amino acid fluxes were statistically different (aspartate, serine, and histidine; P < 0.05), as confirmed by a higher transfer constant. CONCLUSION: Our results indicate that the isolated perfused guinea pig liver is a suitable model for the study of hepatic metabolism.