Rat Liver Surface Research Articles

Development of a Two-Layered Sheet Formulation of 5-Fluorouracil for Application to Rat's Livers to Ensure Controlled Release and Local Drug Disposition.

This study aimed to develop a new and effective application form for the liver surface. We designed a two-layered sheet for the controlled release and local disposition of the anticancer drug, 5-fluorouracil (5-FU), without leakage into the peritoneal cavity. We employed poly(lactic-co-glycolic acid) (PLGA) and hydroxypropyl cellulose (HPC) to form two-layered sheets by attaching a cover sheet and a drug-containing sheet. The prepared two-layered sheets released 5-FU constantly for up to 14 d without any significant leakage from the cover side in vitro. Furthermore, we have applied sheets containing 5-FU to the rat liver surface in vivo. Notably, 5-FU could be detected in the liver attachment region even 28 d after application. The distribution ratio of 5-FU in the attachment region compared to the other liver lobes varied among the sheet formulations with different additive HPC compositions. The area under the liver concentration-time curve (AUC) of 5-FU in the attachment region from 0 to 28 d was the highest in the case of HPC 2% (w/w). This is probably due to the enhanced 5-FU released amount and controlled absorption rate from the liver surface by released HPC. No critical toxic effects were evident by the application of the two-layered sheets from the body weight change and alanine aminotransferase/aspartate aminotransferase (ALT/AST) activities. Consequently, the possible advantage of the two-layered sheets for prolonged retention of a drug in a specific region in the liver was clarified.

The Liver Surface Is an Attractive Transplant Site for Pancreatic Islet Transplantation.

In the current clinical islet transplantation, intraportal transplantation is regarded as the gold-standard procedure. However, in this procedure, 50 to 70% of the transplanted islets are immediately damaged due to a strong innate immune response based on islet–blood contact. We investigated the transplant efficiency of a novel method of liver surface transplantation using a syngeneic keratinocyte sheet to avoid islet–blood contact. To examine the influence of the keratinocyte sheet, substantial amounts of syngeneic islets (8 IEQs/g) were transplanted on the liver surface of diabetic rats, while marginal amounts of islets (4 IEQs/g) were transplanted via intraportal transplantation to compare the transplant efficiency. Blood glucose, intraperitoneal glucose tolerance, immunohistochemistry, and in vivo imaging findings of the cell sheet were evaluated. The study showed that islet transplantation to the liver surface immediately followed by a syngeneic keratinocyte sheet covering was effective for curing diabetic rats, while no rats were cured in the group without the cell sheet. Notably, islet grafts transplanted via this approach appeared to penetrate into the liver parenchyma. However, the transplant efficiency did not reach that of intraportal transplantation. Further refinements of this approach by introducing mesothelial or fibroblast cell sheets in combination with a preferable scaffold for islet grafts may help to improve the transplant efficiency.

Transplantation of fetal liver tissue coated by ultra-purified alginate gel over liver improves hepatic function in the cirrhosis rat model

In this study, we used a new coating agent, that is, ultra-purified alginate gel (UPAL), for fetal liver tissue transplantation. This study aims to compare the effect of UPAL with the effect of other coating agents on improving the effect of fetal liver tissue transplantation in a liver cirrhosis rat model. Prior to the transplantation of wild-type ED14 fetal liver tissues, various coating agents were separately applied on the liver surface of rats with cirrhosis. Then, we compared the engraftment area, engraftment rate and liver function level of these rats. As a result, coating the liver surface of a cirrhosis rat with UPAL obtained the best effect in terms of engraftment area and engraftment rate of the transplanted liver tissue and in the recovery of liver function compared with control group. Therefore, UPAL coating may serve as a novel strategy for liver organoid transplantation.

Influence of Liver Intoxication by Carbon Tetrachloride or D-Galactosamine on Absorption of Fluorescein Isothiocyanate-Dextran-10 and Other Marker Compounds with Different Molecular Weights from the Rat Liver Surface.

We examined the influence of liver disease on the absorption from the liver surface of fluorescein isothiocyanate (FITC)-dextran 10 (FD-10, MW: 11000) and several marker compounds with different molecular weights. The purpose of this study was to determine the feasibility of liver surface application of macromolecular compounds in the disease state. We used male Wistar rats treated with carbon tetrachloride (CCl4) or D-galactosamine (GAL). FD-10 and other marker compounds were applied to the liver surface using a cylindrical diffusion cell in liver-intoxicated rats. The blood, bile, urine, and the remaining solution in the diffusion cell were collected for assay. FD-10 was absorbed by first-order kinetics from the liver surface in the liver-intoxicated rat models. The calculated rate constant ka values in the normal, CCl4 and GAL groups were 0.000965, 0.00125 and 0.00104 min-1, respectively. Increased absorption of FITC-dextrans in the liver-intoxicated rats was observed. In both CCl4 and GAL groups, an inverse relationship was observed between the molecular weight and ka from the rat liver surface of the marker compounds. The limits of the molecular weight absorbed from the liver surface were extrapolated to be 71200, 135000, and 105000 in the normal, CCl4, and GAL groups, respectively. In conclusion, increased absorbability from the rat liver surface indicates that liver surface application for liver targeting of macromolecules in the diseased state is indeed feasible. Therefore, our findings can support further research on liver surface application of drugs under liver disease.

Development of a Patch-Type Flexible Oxygen Partial Pressure Sensor.

Oxygen concentration in living organisms is one of the important vital indicators in emergency care and bedside medical settings. However, the oximetry method has limitations: the measurement site is limited to the tissue containing blood and the absolute value of oxygen concentration cannot be measured. To overcome these limitations, in this work, we develop a new oxygen sensor that can directly measure the oxygen particle pressure (n}{}ptext{O}_{2}n) on the surface of the body and organs. A light emitting diode (LED) and a photodiode (PD) were embedded in a dimethylpolysiloxane substrate mixed with carbon nanotubes. The effectiveness of the device was evaluated using calibration, bending strain tests, time and frequency response, and finally in vivo assessments. The results reveal that the calibration experiment of the fabricated oxygen sensor device showed high sensitivity. The carbon nanotube electrode has a sufficient bending resistance and does not affect the response characteristics of the LED and PD, that is, it does not affect the oxygen measurement. In vivo assessment shows that the developed patch-type flexible oxygen sensor can accurately measure n}{}ptext{O}_{2}n by attaching it to tissues or organs having irregularities or curved surfaces and actual measurements on rat liver surface demonstrated its feasibility.

Topical Application of Mesenchymal Stromal Cells Ameliorated Liver Parenchyma Damage After Ischemia-Reperfusion Injury in an Animal Model.

Ischemia-reperfusion injury (IRI) is commonly encountered after liver surgery. This study evaluated the hepatoprotective effects of topically applied adipose-derived mesenchymal stromal cells (ADMSCs) on hepatic IRI in a rat model. ADMSCs from transgenic green fluorescent protein Sprague-Dawley rats were topically applied to the liver surface of Sprague-Dawley rats after hepatic IRI and fixed in position by fibrin glue (group A, n = 24). An equivalent amount of ADMSCs were administered through the portal (group B, n = 24) or tail vein (group C, n = 24). In the control group (group D, n = 20), no treatment was given to the IRI liver. All the rats in group A and group D survived. Within 2 days after hepatic IRI, only 50% of rats survived in group B, and ADMSCs were detected in thromboemboli within large vessels. 62.5% of the rats died in group C because most of the ADMSCs were trapped in the lungs. ADMSCs migrated across the liver capsule and homed to the injured liver parenchyma 3 days after topical application in group A. The homed ADMSCs expressed hepatocyte nuclear factor-4α and hepatocyte nuclear factor-1. Compared with group D, the rate of hepatic regeneration in group A was enhanced with less inflammation, smaller necrotic areas, and improved liver function. Proinflammatory cytokines IL-6, IL-21, and CD70 were significantly downregulated in group A by 6.3-, 2.7-, and 12.7-fold, respectively (P < 0.05). The neurogenic locus NOTCH homolog protein pathway was activated in the topical ADMSCs. Topically applied adipose-derived mesenchymal stromal cells demonstrated hepatoprotective effects on hepatic IRI in an animal model.

Influence of vasomodulators and tumor transplantation on the disposition of 5-fluorouracil after application to the liver surface in rats.

This study investigated the effect of epinephrine (a vasoconstrictor) and hydralazine (a vasodilator) on the hepatic disposition of 5-fluorouracil (5-FU) after application to the surface of the liver in rats. Normal livers were compared with a Walker 256 carcinoma cell tumor model. A cylindrical diffusion cell was attached to the liver surface. 5-Fluorouracil was added into the diffusion cell in combination with vasomodulators or after pretreatment with epinephrine. After selected treatment times, the 5-FU concentrations were assayed at three sites in the excised livers. The 5-FU concentration in the region under the cell attachment site (site 1) was significantly higher after concomitant application of 5-FU and epinephrine, compared with 5-FU alone, and increased in an epinephrine dose-dependent manner. On the other hand, preferential distribution of 5-FU at site 1 was not seen when applied in combination with hydralazine. After 10min of epinephrine pretreatment, the concentration of 5-FU at site 1 was approximately two times higher than that for the control. Furthermore, the 5-FU concentration at site 1 of the tumor model was greatly increased compared with the normal liver. These results suggest that application of epinephrine to the liver surface might enhance the accumulation of 5-FU at the desired target site.

Effect of Metabolic Inhibitors on the Hepatic Disposition of 5-Fluorouracil after Application to the Rat Liver Surface.

We evaluated the effects of 5-fluorouracil (5-FU) metabolic inhibitors, gimeracil or uridine, on the hepatic disposition of 5-FU after application to the liver surface in rats, aiming to enhance the availability of 5-FU in the liver. 5-FU solution with or without metabolic inhibitors was applied to the rat liver surface using a cylindrical diffusion cell. The liver, blood and the remaining solution in the diffusion cell were collected at specified times, and assayed for 5-FU content. 5-FU absorption properties were not altered by addition of gimeracil and uridine. The 5-FU concentration in the diffusion cell attachment site of the rat liver (site 1) at 0.1-0.4 M ratios of gimeracil to 5-FU was significantly higher than that of the control. On the contrary, the addition of uridine did not increase the 5-FU concentration at site 1. At a 0.1 M ratio of gimeracil to 5-FU, the maximum 5-FU plasma concentration was the lowest, and the area under the 5-FU concentration-time curve at site 1 was 3.4 times greater than that of the control. We demonstrated that applying 5-FU with gimeracil to the rat liver surface could increase the availability of 5-FU in the liver.

Effect of absorption enhancers on the absorption of FD-4 as a poorly absorbable marker macromolecule from the liver surface in rats

The purpose of this study is to improve the rate at which drugs are absorbed from the liver surface by utilizing absorption enhancers such as extracted saponin from Saponaria officinalis (saponin), sodium caprate (Na-Cap), sodium salicylate (Na-Sal), and disodium ethylenediamine-tetraacetic acid. Fluorescein isothiocyanete-dextran with molecular weights of 4400 (FD-4) as a poorly absorbable marker macromolecule was applied to the rat liver using a cylindrical diffusion cell, in the presence or absence of the absorption enhancers. After a 15-min pre-treatment with or the co-administration of saponin or Na-Cap, the absorption ratios of FD-4 and plasma area under the curve were significantly increased, compared to control values. On the other hand, the absorption of FD-4 was increased by a 30-min pre-treatment with Na-Sal. The absorption of FD-4 applied to the rat liver was promoted by absorption enhancers. This approach could have therapeutic use for poorly absorbable compounds.

Anti-fibrotic effects of fresh and cryopreserved human amniotic membrane in a rat liver fibrosis model

The human amniotic membrane (hAM), thanks to its favorable properties, including anti-inflammatory, anti-fibrotic and pro-regenerative effects, is a well-known surgical material for many clinical applications, when used both freshly after isolation and after preservation. We have shown previously that hAM patching is a potential approach to counteract liver fibrosis. Indeed, when fresh hAM was used to cover the liver surface of rats with liver fibrosis induced by the bile duct ligation (BDL) procedure, the progression and severity of fibrosis were significantly reduced. Since cryopreservation enables safety and long-term storage of hAM but may influence its functional properties, here we compared the anti-fibrotic effects of fresh and cryopreserved hAM in rats with BDL-induced liver fibrosis. After BDL, the rat liver was covered with a piece of fresh or cryopreserved hAM, or left untreated. Six weeks later, the degree of liver fibrosis was assessed histologically using the Knodell and the METAVIR scoring systems. Digital image analysis was used to quantify the percentage of the areas of each liver section displaying ductular reaction, extracellular matrix (ECM) deposition, activated myofibroblasts and hepatic stellate cells (HSCs). Liver collagen content was also determined by spectrophotometric technique. The degree of liver fibrosis, ductular reaction, ECM deposition, and the number of activated myofibroblasts and HSCs were all significantly reduced in hAM-treated rats compared to control animals. Fresh and cryopreserved hAM produced the same anti-fibrotic effects. These findings indicate that cryopreservation maintains the anti-fibrotic properties of hAM when used as a patch to reduce the severity of liver fibrosis.

Effect of viscous additives on the absorption and hepatic disposition of 5-fluorouracil (5-FU) after application to liver surface in rats

Objectives The aim was to study the effect of viscous additives on the absorption and hepatic disposition of 5-fluorouracil (5-FU) after application to the liver surface in rats. Methods 5-FU solution with or without viscous additives was applied to the rat liver surface with a cylindrical diffusion cell. Then, blood and the remaining solution in the diffusion cell were collected at selected times, followed by excision of the liver. The excised liver was divided into three sites and assayed for 5-FU content. Key findings The absorption rate of 5-FU from the liver surface was decreased in the presence of carboxymethylcellulose sodium (CMC-Na) and polyvinyl alcohol (PVA) as compared with the control. The k(a) values of PVA 15% and CMC-Na 1% were reduced to about 80 and 67% of the control. The maximum plasma concentration of 5-FU was decreased by incorporation of viscous additives. The 5-FU concentration at the diffusion cell attachment site of the liver (site 1) plateaued at 180 min in the absence of viscous additives. On the other hand, the concentration of 5-FU at site 1 increased in a time-dependent manner until 360 min in the presence of viscous additives. Conclusion Viscous additives might be useful for retaining drugs at their application site and controlling the rate of absorption from the liver surface.

Mechanism for drug absorption from rat-liver surface membrane: effect of dose and transport inhibitors on the pharmacokinetics of phenol red.

We examined the effect of dose and transport inhibitors on the pharmacokinetics of phenol red as a model drug after application to rat liver surface in-vivo, employing a cylindrical glass cell (i.d. 9 mm, area 0.64 cm2), to elucidate the mechanism for drug absorption from liver surface membrane. Absorption ratios of phenol red in 6 h were determined to be 91.1, 91.8 and 89.9% at a dose of 0.3, 1 and 3 mg, respectively. The AUC value for plasma concentration profile of phenol red was proportional to the dose. It is thus suggested that the absorption process of phenol red from rat liver surface does not approach saturability. The time course of the remaining amount of phenol red in the glass cell obeyed first-order kinetics at a dose of 0.3 mg, and its rate constant Ka was calculated to be 0.0069 min-1. Moreover, no significant difference was seen in the Ka value within the dose range of 0.3-3 mg, which was estimated by curve fitting of the plasma concentration profile of phenol red after application to rat liver surface in the two-compartment model with first-order absorption. 2,4-Dinitrophenol (0.3 mg) and probenecid (0.5 and 1 mg), inhibitors of metabolic energy and anion transport, respectively, had no significant effect on the pharmacokinetics of phenol red after application to rat liver surface. These data demonstrate that a specific transport mechanism such as active transport is not involved in phenol red absorption from rat liver surface membrane.

Effect of application volume and area on the absorption of phenol red, as a model drug, from the liver surface in rats.

To determine the influence of the method of administration of a pharmaceutical formulation we have examined the importance of application volume and area in the absorption of phenol red, as a model drug, from the rat-liver surface. When 1 mg phenol red was applied to the rat-liver surface, in-vivo, in three volumes (0.1, 0.2 or 0.334 mL) using a cylindrical glass cell (i.d. 9 mm), the shape of the plasma concentration profile differed greatly, particularly the maximum concentration. These patterns were well fitted by a two-compartment model with first-order absorption, and the absorption-rate constant Ka obtained was inversely proportional to the application volume. The absorption ratio and biliary recovery of phenol red after 6 h increased with glass cell area (i.d. 6, 9 or 14 mm; area 0.28, 0.64 or 1.54 cm2). Furthermore, the permeability coefficient Papp derived from Ka did not depend on application area, indicating no difference in the absorption characteristics of the liver surface. This also implies transport of the drug by passive diffusion from the liver surface. After intraperitoneal administration to the rat-liver surface for clinical application, increasing the application volume resulted in the delayed disappearance of phenol red from the plasma. However, the difference was not as marked as that obtained by use of the glass cell. The assumption that the effective area relating to the absorption changed with the application volume enabled us to estimate Papp. Consequently, we speculate that absorbability can be estimated precisely by consideration of application volume and area.

Absorption of organic anions as model drugs following application to rat liver surface in-vivo.

Absorption of organic anions (phenol red, bromphenol blue and bromosulphonphthalein) has been studied after their application to rat liver surface in-vivo, employing a cylindrical glass cell (i.d. 9 mm, area 0.64 cm2). Each drug appeared gradually in the blood with the peak level at about 1 h, after which its concentration declined slowly. Absorbed model drug was efficiently excreted into the bile. These observations appear to indicate the possibility of drug absorption from liver surface membrane. Absorption of model drugs was estimated to be more than 59% in 6 h. The biliary recovery and metabolism of phenol red did not change as compared with that after intravenous administration.

Relationship between lipophilicity and absorption from the liver surface of paraben derivatives and antipyrine in rats

The importance of drug lipophilicity on absorption from the liver surface was examined in rats using paraben derivatives, antipyrine, Sudan III, and Sudan blue. The log partition coefficient (PC) of n-octanol/water ranged from -1.39 to 4.62. The compounds were applied to the rat liver surface using a cylindrical diffusion cell (i.d. 9 mm). The rate of absorption at 15 min was calculated to be 13.9% for paraben, much lower than that for its derivatives methylparaben, propylparaben and butylparaben (∼ 80%). The obtained first-order absorption rate constant (k(a) ) of paraben, methylparaben, propylparaben and antipyrine increased according to lipophilicity. Further lipophilicity resulted in a fall in k(a) , implying the importance of affinity for lipids and water in absorption from the liver surface. Thus, a compound with a log PC of around 2.5 is considered to have maximum absorbability from the rat liver surface. A good relationship (r(2) = 0.97) was recognized between the log k(a) and log reciprocal value with the square root of molecular weight of the compounds with a log PC below 2.5. The rate of absorption of a drug from the liver surface could be estimated from physicochemical properties such as lipophilicity and molecular weight.

Effect of instillation method on the absorption of phenolsulphonphthalein as a model drug from the liver and small intestinal serosal surface in rats.

We have examined the effect of the instillation method on the absorption of a drug from the liver and the small intestinal serosal surface in rats. We performed continuous microinstillation via an infusion pump and bolus instillation via a syringe, using phenolsulphonphthalein (phenol red) as the model drug. After continuous microinstillation of phenolsulphonphthalein 2.35 mg in 235 microL for 5 min on the liver and small intestinal serosal surface in rats, the AUC (area under the curve) of the plasma concentration profile up to 60 min was significantly higher compared with bolus instillation. A similar trend was observed after continuous microinstillation of phenolsulphonphthalein 2.35 mg in 117.5 microL for 2.5 min. The calculated absorption rate constants (Ka) after continuous microinstillation of phenolsulphonphthalein based on a two-compartment model with first-order absorption were higher than those after bolus instillation on the liver and small intestinal serosal surface at either instillation concentration. Moreover, Ka was increased after continuous microinstillation of 2.35 mg in 117.5 microL at either instillation site. Instillation of phenolsulphonphthalein on the liver surface resulted in a 1.2- to 2.3-fold higher Ka compared with the small intestinal serosal surface. This tendency was marked after continuous microinstillation of 2.35 mg in 117.5 microL. In conclusion, absorption could be enhanced by instilling a small amount of drug solution on the liver surface gradually and continuously, suggesting a promising approach for instillation site-selective drug delivery in the peritoneal cavity.

Development of Drug Delivery System by Utilizing Absorption from Liver Surface and Its Application

Because it is difficult to achieve local drug activity following administration by the conventional intravenous and oral routes, I sought to develop a new route of administration utilizing drug absorption from the liver surface in order to target that organ. Although direct application to the liver surface should yield local drug distribution, drug absorption from the liver surface has not been reported in the literature. Therefore, we analyzed, as a model, the efficiency of absorption of several organic anions and dextrans of various molecular weights following application to the rat liver surface in vivo using a cylindrical diffusion cell. Each compound appeared gradually in the plasma, followed by excretion into the bile and/or urine, indicating the possibility of drug absorption from the liver surface. The absorption process from the liver surface may not involve a specific transport system because dose and transport inhibitors had no detectable effect. In addition, molecular weight was found to be a determinant of absorption through the liver surface. The efficiency of targeting desired region in the liver was enhanced considerably by application to the liver surface, compared to intravenous administration. Moreover, I have obtained several promising results from the application of this new drug delivery system to anticancer drugs and gene therapy. On the other hand, I have also clarified the characteristics of drug absorption from the surfaces of the kidney, stomach, cecum and small intestine, and plan to apply the physiological findings to other fields.

Absorption and Distribution Characteristics of 5-Fluorouracil (5-FU) after an Application to the Liver Surface in Rats in Order to Reduce Systemic Side Effects

The present study was undertaken to elucidate the absorption and distribution characteristics of 5-fluorouracil (5-FU) after its application to the liver surface in rats to examine the possibility of reducing the systemic side effects of this agent. 5-FU was applied to the surface of the liver by employing a cylindrical diffusion cell. Approximately 69% of the dose was absorbed in 360 min. The time course of the change in the amount of 5-FU remaining in the diffusion cell obeyed first-order kinetics. Also, a linear relationship was observed between the apparent permeability coefficient, P app, and the reciprocal of the square root of the molecular weight of several compounds including 5-FU. The estimated P app value of 5-FU was in good agreement with the experimental value. The plasma concentration of 5-FU was low (<1.2 microg/ml) until 360 min after the application. Following i.v. administration, 5-FU was rapidly eliminated from the plasma and could not be detected at 120 min. In the analysis of tissue distribution, the liver was divided into three sites; the region under the diffusion cell attachment site (site 1), the treated lobe excluding site 1 (site 2), and untreated lobes (site 3). After being administered i.v., 5-FU mainly distributed in the kidney, and the concentration in the liver was significantly lower than that in kidney, spleen, or heart. After its application to the liver surface, however, 5-FU preferentially distributed at site 1, and was not detected at the other sites or in other tissues. Thus, these results suggested the possibility of a reduction in the systemic side effect of 5-FU on its application to the liver surface.

Regional Delivery of Model Compounds and 5-Fluorouracil to the Liver by Their Application to the Liver Surface in Rats: Its Implication for Clinical Use

The purpose of this study was to examine drug distribution in the liver after drug application to the rat liver surface. Phenolsulfonphthalein (PSP) and fluorescein isothiocyanate dextran (MW 4400, FD-4) as model compounds or 5-fluorouracil (5-FU) was applied to the rat liver surface by employing a cylindrical diffusion cell (i.d. 9 mm, 0.64 cm2). Then, blood and the remaining solution in the diffusion cell were collected at selected times, followed by excision of the liver. The excised liver was divided into three sites: the region under the diffusion cell attachment site (site 1), the applied lobe except for site 1 (site 2), and non-applied lobes (site 3). In the case of i.v. administration, there were no differences in PSP concentrations among the three sites of the rat liver, and the concentrations rapidly decreased. On the other hand, the PSP concentration in site 1 after application to the rat liver surface was considerably higher than in site 2 and site 3. In addition, the area under the curve (AUC) value (AUCsite1), calculated from the PSP concentration profile in site 1, was about 10 times larger than that in site 3. A similar trend of regional delivery advantage by liver surface application was observed in the case of the macromolecule model FD-4, with a marked AUCsite1 of about 5 times larger than the other two sites. Moreover, we clarified that the anticancer drug 5-FU preferentially distributed in site 1 after application to the rat liver surface. These results demonstrate the possibility of regional delivery of drugs to the liver by application to the liver surface.

Bonghan Ducts on the Surface of Rat Liver(International Conference on Mind Body Science : Physical and Physiological Approach joint with The Eighteenth Symposium on Life Information Science)

Bonghan Ducts on the Surface of Rat Liver(International Conference on Mind Body Science : Physical and Physiological Approach joint with The Eighteenth Symposium on Life Information Science)