Biomedical subjects

G Khang

Publications and source records attributed to G Khang.

At least 19 recordsLinked to original sources

PubMed2002

Gradual potentiation of isometric muscle force during constant electrical stimulation.

An investigation was carried out into how stimulation frequency and stimulation history affect the potentiation of muscle force during 20s of constant stimulation of the two knee extensors in isometric conditions. Stimulation frequency significantly affected the potentiation pattern: low-frequency (2.5-10 Hz) stimulation showed a reduction and subsequent enhancement of force, and high-frequency (14.3-25 Hz) stimulation showed only enhancement of force. The degree of enhancement in force and time-to-peak decreased with the stimulation frequency. Whereas 40 Hz conditioning stimulation enhanced the muscle force, 14 Hz stimulation after 10s of rest induced little force enhancement (8% in both muscles). When the frequency of the conditioning stimulation was 14 Hz and the main 14 Hz stimulation was applied after 50s of rest, the initial force at the main stimulation was similar to the final force value of the conditioning stimulation (above 90% similarity). The potentiated twitch force slowly decayed during rest, with an average time constant of 2.4 min. These observations indicate that muscle potentiation depends on the stimulation frequency and stimulation history, and therefore a computer model of potentiation can play an important role in predicting muscle force and body movement induced by electrical stimulation.

Adult↗

PubMed2001

Quantitative analysis of fentanyl in rat plasma by gas chromatography with nitrogen-phosphorus detection.

A sensitive assay method was developed to determine fentanyl, an opiate agonist, in rat plasma by gas chromatography with nitrogen-phosphorus detection. For the pretreatment of plasma samples, sodium hydroxide was added to denature protein and n-butyl chloride was used to extract fentanyl. The calibration curve was linear within the concentration range 0.5 to 50 ng/ml (r=0.9997). The limit of detection was 0.1 ng/ml, and 0.5 ng/ml could be quantified with acceptable precision. Furthermore, fentanyl could be determined in only 200 microl of rat plasma. The method has been successfully applied to an intramuscular pharmacokinetic study at a dose of 10 microg/kg. Therefore, the current method is a valuable analytical tool for investigating the pharmacokinetics of fentanyl at low clinical doses.

Analgesics, Opioid↗

PubMed2001

Preparation and characterization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) microspheres for the sustained release of 5-fluorouracil.

Biodegradable microspheres were prepared with poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV, 85:15 by mole ratio of hydroxybutyrate to hydroxyvalerate) by an water-in-oil-in-water (W/O/W), oil-in-water (O/W) and oil-in-oil (O/O) solvent evaporation method for the sustained release of anti-cancer drug, 5-fluorouracil (5-FU) with controlling the fabrication conditions. The shape of microspheres prepared was relatively rough due to highly crystalline property of PHBV and spherical. The efficiency of 5-FU loading into the PHBV microsphere with O/O method was over 80% compared to that 7% for microspheres by O/W method and below 1% for microspheres by a conventional W/O/W method. However, the most desirable release pattern can be achieved from the O/W method due to the cosolvent effect. The effects of preparation conditions such as the type and amount of surfactant, initial amount of loaded drug, the temperature of solvent evaporation, and etc. on the morphology for W/O/W method were investigated. Possible mechanisms of the desirable sustained release pattern for O/W system have been proposed.

Antineoplastic Agents↗

PubMed2000

The Effect of Fluid Shear Stress on Endothelial Cell Adhesiveness to Polymer Surfaces with Wettability Gradient.

In this study, the adhesive strength of endothelial cells (ECs) attached on polymer surfaces with different hydrophilicity was investigated using wettability gradient polyethylene (PE) surfaces prepared by corona discharge treatment from a knife-type electrode whose power increases gradually along the sample length. The EC-attached wettability gradient surfaces were mounted on parallel-plate flow chambers in a flow system prepared for cell adhesiveness test. Three different shear stresses (150, 200, and 250 dyne/cm(2)) were applied to the flow chambers and each shear stress was maintained for 120 min to investigate the effect of shear stress and surface hydrophilicity on the EC adhesion strength. It was observed that the ECs were adhered more onto the positions with moderate hydrophilicity of the wettability gradient surface than onto the more hydrophobic or hydrophilic positions. The maximum adhesion of the cells appeared at around water contact angles of 55 degrees. The EC adhesion strength was higher on the hydrophilic positions than on the hydrophobic ones. However, the maximum adhesion strength of the cells also appeared at around water contact angles of 55 degrees. More than 90% of the adhered cells remained on that position after applying the shear stress, 250 dyne/cm(2) for 2 h, whereas the cells were completely detached on the hydrophobic position (water contact angle, about 86 degrees ) within 10 min after applying the same shear stress. It seems that surface hydrophilicity plays a very important role for cell adhesion strength. Copyright 2000 Academic Press.

Journal Article↗

PubMed2000

Nifedipine controlled delivery by sandwiched osmotic tablet system.

The sandwiched osmotic tablet system (SOTS), which is composed of a sandwiched osmotic tablet core surrounded by a cellulose acetate membrane with two orifices on both side surfaces, has been successfully prepared with the purpose of delivering nifedipine. The sandwiched osmotic tablet core consists of a middle push layer and two attached drug layers. Influences of tablet formulation variables, orifice size and membrane variables on nifedipine release of SOTS have been studied. It was found that potassium chloride amount of push layer and polyethylene oxide amount of drug layer had markedly positive effects on nifedipine release. A push layer/drug layer co-controlled osmotic delivery mechanism and the optimal core formulation have been proposed. The appropriate orifice size was observed in the range of 0.50-1.41 mm. It was also found that the drug release rate of SOTS could be increased by incorporating hydrophilic plasticizer in the membrane, whereas it decreased with hydrophobic plasticizer. It has been observed that the SOTS gives fairly comparable in vitro release features as that of commercialized push-pull osmotic tablet system, such as an approximately constant rate up to 24 h and independence of release media and agitation rate. Exempting side identification before drilling, it is easier to prepare the SOTS than the push-pull osmotic tablet system.

Calcium Channel Blockers↗

PubMed2000

Monolithic osmotic tablet system for nifedipine delivery.

The monolithic osmotic tablet system, which is composed of a monolithic tablet coated with cellulose acetate (CA) membrane drilled with two orifices on both side surfaces, has been described. The influences of tablet formulation variables including molecular weight (MW) and amount of polyethylene oxide (PEO), amount of potassium chloride (KCl), and amount of rice starch as well as nifedipine loading have been investigated. The optimal tablet formulation and the osmotic-suspending co-controlled delivery mechanisms have been proposed. Orifice size and membrane variables including nature and amount of plasticizers as well as thickness on drug release have also been studied. The in vitro release profiles of the optimal system have been evaluated in various release media and different agitation rates, and compared with commercialized conventional capsule and push-pull osmotic tablet. It was found that PEO with MW of 300000 g/mol was suitable to be thickening agent, both amount of KCl and amount of PEO had comparable and profoundly positive effects, while nifedipine loading had a strikingly negative influence on drug release. It could be found that the optimal orifice size was in the range of 0.25-1.41 mm. It has also been observed that hydrophilic plasticizer polyethylene glycol (PEG) improved drug release, whereas hydrophobic plasticizer triacetin depressed drug release when they were incorporated in CA membrane. The monolithic osmotic tablet system was found to be able to deliver nifedipine at the rate of approximate zero-order up to 24 h, independent of both environmental media and agitation rate, and substantially comparable with the push-pull osmotic tablet. The monolithic osmotic tablet system was simple to be prepared as exempting from push layer and simplifying in the orifice drilling compared with the push-pull osmotic tablet. The monolithic osmotic tablet system may be used in drug controlled delivery field, especially suitable for water-insoluble drugs.

Calcium Channel Blockers↗

PubMed1999

The effect of the chemical structure of the phospholipid polymer on fibronectin adsorption and fibroblast adhesion on the gradient phospholipid surface.

The interaction between biocomponents and the polyethylene (PE) surface modified with poly[omega-methacryloyloxyalkyl phosphorylcholine (MAPC)] was considered taking into account the surface characteristics, i.e., density, mobility, and orientation of the poly(MAPC). The PE surface, grafted gradually with the poly(MAPC) was prepared by corona irradiation method. The amount of peroxide produced on the PE surface which was determined with 1,1-diphenyl-2-picryl-hydrazyl, increased with an increase in the energy of the corona. The surface density of the poly(MAPC) was increased with an increase in the amount of the peroxides produced by the corona irradiation. The orientation and mobility of the poly(MAPC) grafted on the PE surface was evaluated with 1,6-diphenyl-1,3,5-hexatriene. The orientation of the poly[6-methacryloyloxyhexyl phosphorylcholine (MHPC)] which has six methylene chains between the phospholipid polar group and the backbone was higher than that of other poly(MAPC)s. The mobility of the poly(MAPC) decreased with an increase in the methylene chain length in the MAPC unit. The fibronectin adsorption on the gradient PE sheet grafted with poly(MAPC) was determined with enzyme-labeled immunoassay. The amount of adsorbed fibronectin on the PE grafted with poly[2-methacryloyloxyethyl phospohorylcholine(MPC)] and poly(MHPC) decreased with an increase in their surface density. Especially, the PE sheet grafted with the poly(MHPC) was effectively reduced compared with other poly(MAPC)s. On the poly[10-methacryloyloxydecyl phosphorylcholine (MDPC)], there is a minimum amount of adsorbed fibronectin. The fibronectin adsorption pattern on the PE sheet grafted with poly(MAPC) was quite different from the chemical structure of the MAPC unit. The human normal diploid fibroblasts (WI-38 cells) were cultured on the gradient PE sheet grafted with poly(MAPC) changing the concentration of seeded WI-38 cells. The adhesion behavior of the WI-38 cells was different depending on the concentration of the seeded WI-38 cells. When the concentration was low, the number of the adherent WI-38 cells had the same tendency as fibronectin adsorption. The gradient PE sheet grafted with the poly(MHPC) effectively reduced WI-38 cells adhesion even when the concentration of the WI-38 cells was high. The biocompatibility of polymer surfaces can be improved by highly oriented phosphorylcholine group.

Adsorption↗

PubMed1999

Interaction of fibroblasts on polycarbonate membrane surfaces with different micropore sizes and hydrophilicity.

Surface topography appears to be an important but often neglected factor in implant performance. In this study, fibroblasts were cultured on a range of porous polycarbonate (PC) membranes with well defined surface topography (track-etched micropores, 0.2-8.0 microm in diameter) and wettability gradients. The wettability gradient on the PC membrane surfaces was produced by treating the surfaces with corona from a knife-type electrode whose power increased gradually along the sample length. The PC membrane surfaces were characterized by scanning electron microscopy (SEM) and the water contact angle measurement. Fibroblasts were cultured on the corona-treated PC membrane surfaces with different micropore sizes for 1 and 2 days. The cells attached on the membrane surfaces were examined by SEM and the cell density on the surfaces was estimated by counting the number of attached cells along the wettability gradient. It was observed that the cells were adhered and grew more on the hydrophilic positions of the membrane surfaces than the more hydrophobic ones, regardless of micropore size. It was also observed that cell adhesion and growth decreased gradually with increasing micropore size of the membrane surfaces. It seems that the cell adhesion and growth were progressively inhibited as the membrane surfaces had micropores with increasing size, probably due to surface discontinuities produced by tract-etched pores. On the membrane surfaces with smaller micropore sizes, the cells seemed to override these surface discontinuities.

3T3 Cells↗

PubMed1999

Preparation and characterization of Japanese encephalitis virus vaccine loaded poly(L-lactide-co-glycolide) microspheres for oral immunization.

Japanese encephalitis virus (JEV) vaccine loaded biodegradable poly(L-lactide-co-glycolide) (PLGA) microspheres (MSs) were prepared by W/O/W solvent evaporation method to study the possibility for oral vaccination. The influence of several preparation parameters as stirring rate, types and concentration of emulsifier, PLGA concentration, etc. has been observed on size, size distribution and biodegradation. The mean MSs size decreased when the agitation speed and the concentration of emulsifier were increased, and when the PLGA concentration was decreased. The surface morphology of porous and nonporous JEV vaccine loaded PLGA MSs was prepared from polyvinylalcohol and sodium dodecyl sulfate as used emulsifiers, respectively. From the assay of lactic acid and scanning electron microscope observation, it can be suggested that the rate of biodegradation of nonporous MSs was slower than that of porous surface morphology due to lower the surface area. Mechanisms of the formation of porous and nonporous surface by different types of emulsifier, and the biodegradation of MSs have been proposed. Also, the size and size distribution of JEV vaccine loaded PLGA MSs were discussed to apply oral vaccination through the Peyer's patches across the gastrointestinal tract.

Absorption↗

PubMed1999

Interaction of fibroblast cells on poly(lactide-co-glycolide) surface with wettability chemogradient.

Chemogradient surfaces whose properties are changed gradually along the sample length are of particular interest for the basic studies of the interaction between biological species and surfaces since the effect of a selected property can be examined in a single experiment on one surface. A wettability chemogradient on the poly(L-lactide-co-glycolide) (PLGA) films by treating them in air with corona from a knife-type electrode whose power increases gradually along the sample length. The PLGA surfaces oxidized gradually with the increasing corona power, and the wettability chemogradient was created on the surfaces as evidenced by the measurement of water contact angles and electron spectroscopy for chemical analysis. The wettability chemogradient PLGA surfaces prepared were used to investigate the interaction of fibroblast cells in terms of the surface hydrophilicity/hydrophobicity of PLGA surface. The cells adhered and grown on the chemogradient surface along the sample length were counted and observed by scanning electron microscopy. It was observed that the cells were adhered, spread, and grown more onto positions with moderate hydrophilicity of the wettability chemogradient PLGA surface than onto the more hydrophobic or hydrophilic positions. The maximum adhesion and growth of the fibroblast cells appeared at around water contact angle of 55 degrees. It seems that the wettability plays important roles for cell adhesion, orientation, spreading and growth on the PLGA surface. It might be that this surface modification technique can be used for improving the adhesion and growth of cell onto PLGA film and scaffolds, and can be applicable in the area of the tissue engineering.

3T3 Cells↗

PubMed1999

Sandwiched osmotic tablet core for nifedipine controlled delivery.

The sandwiched osmotic tablet core, which is composed of a middle push layer and two attached drug layers, has been prepared and systematically studied with the purpose of delivering water-insoluble nifedipine. The advantage of the sandwiched osmotic tablet system over the commercialized push-pull osmotic tablet system is its simplicity of preparation, as the surface identification was avoided. It was observed that polyethylene oxides (PEO) with molecular weight (MW) of 300,000 and 8,000,000 g/mole were suitable for the thickening agent of drug layer and the expandable hydrogel of push layer, respectively. The weight ratio of 190/190 for drug layer/push layer was also found to be suitable. It has been observed that PEO amount of the push layer and the KCl amount of the drug layer had profoundly positive influence on nifedipine release. A push layer/drug layer co-controlled osmotic delivery mechanism has been proposed and the optimal tablet formulation has been obtained. It was also found that PEO and nifedipine were miscible, which may support the application of PEO in nifedipine dosage forms. Meanwhile, the PEO/nifedipine binary phase diagram has been constructed. The sandwiched osmotic tablet system can deliver nifedipine in an approximate zero-order rate up to 24 hours. It may be potentially used for the delivery of water-insoluble drugs.

Calcium Channel Blockers↗

PubMed1998

Interaction of Different Types of Cells on Polymer Surfaces with Wettability Gradient.

Gradient surfaces whose properties are changed gradually along the sample length are of particular interest for basic studies of the interaction between biological species and surfaces since the effect of a selected property can be examined in a single experiment on one surface. We prepared a wettability gradient on low density polyethylene (PE) sheets by treating them in air with the corona from a knife-type electrode whose power increases gradually along the sample length. The PE surfaces oxidized gradually with the increasing corona power, and the wettability gradient was created on the surfaces as evidenced by the measurement of water contact angles, Fourier transform infrared spectroscopy in the attenuated total reflectance mode, and electron spectroscopy for chemical analysis. The wettability gradient surfaces prepared were used to investigate the interaction of different types of cells (Chinese hamster ovary, fibroblast, and endothelial cells) as well as serum proteins in terms of the surface hydrophilicity/hydrophobicity of polymeric materials. The cells adhered and grown on the gradient surface along the sample length were counted and observed by scanning electron microscopy. It was observed that the cells were adhered, spread, and grown more onto the positions with moderate hydrophilicity of the wettability gradient surface than onto the more hydrophobic or hydrophilic positions. The maximum adhesion and growth of the cells appeared at around water contact angles of 55 degrees, regardless of the cell types used. This result seems closely related to the serum protein adsorption on the surfaces; the serum proteins were also adsorbed more onto the positions with moderate hydrophilicity of the wettability gradient surface. Copyright 1998 Academic Press.

Journal Article↗

PubMed1998

Platelet adhesion onto chargeable functional group gradient surfaces.

Functional group gradients were prepared on low-density polyethylene (PE) sheets. The surface density of grafted functional groups was gradually changed along the sample length by way of corona discharge treatment with gradually increasing power following graft copolymerization of acrylic acid (AA), sodium p-styrene sulfonate (NaSS), or N,N-dimethyl aminopropyl acrylamide (DMAPAA). AA and NaSS are negatively chargeable and DMAPAA is positively chargeable in phosphate-buffered saline or plasma solution at pH 7.3-7.4. The prepared functional group gradient surfaces were characterized by measurement of the water contact angle, by electron spectroscopy for chemical analysis, and by Fourier transform infrared spectroscopy in the attenuated total reflectance mode. All these measurements indicated that the functional groups were grafted onto the PE surfaces with gradually increasing density. The platelets adhered to the functional group gradient surfaces along the sample length were counted and observed by scanning electron microscopy. It was observed that the platelet adhesion to the gradient surfaces decreased gradually with the increasing surface density of functional groups. This may be related to the hydrophilicity of the surfaces. The DMAPAA-grafted surface showed a large amount of platelet adhesion, probably due to its positive charge character, while the AA-grafted surface, which is charged negatively, showed poor platelet adhesion. However, the NaSS-grafted surface, which is also charged negatively, showed a relatively large amount of platelet adhesion. This may be associated with the existence of an aromatic ring close to the ionizable group in NaSS. It seems that surface functional groups and their charge character, as well as wettability, play important roles for platelet adhesion.

Acrylates↗

PubMed1998

Platelet adhesion on the gradient surfaces grafted with phospholipid polymer.

We have synthesized omega-methacryloyloxyalkyl phosphorylcholine (MAPC) polymers as new blood-compatible materials, with attention to the surface structure of the biomembrane and investigated their blood compatibility. The blood compatibility observed on the MAPC polymers is due to their strong affinity to phospholipids. When the blood comes in contact with the MAPC polymer, phospholipids in the plasma preferentially adsorb on the surface, compared with the plasma proteins or cells. The adsorbed phospholipids construct a biomembrane-like structure on the MAPC polymer surface. The MAPC polymers then have an excellent blood compatibility. In this study, we prepared a gradient poly(MAPC)-grafted polyethylene (PE) surface using a corona discharge treatment method to clarify the effect of the chemical structure of the MAPC unit on the blood compatibility of the MAPC polymers. The surface composition of MAPC and the hydrophilicity on the poly(MAPC)-grafted PE surface were determined by X-ray photoelectron spectroscopic (XPS) analysis and contact angle measurement with water, respectively. The phosphorus/carbon (P/C) ratio determined by the XPS analysis increased, but the water contact angle decreased with increasing corona irradiation energy. These results indicated that the surface density of the MAPC unit was increased. More than 2.5 cm from the starting point of the corona irradiation, the P/C ratio and water contact angle of the surface achieved a constant level. Thus, the surface was completely covered with the grafted poly(MAPC) chain. The effect of the methylene chain length of the MAPC unit on surface properties was also observed. The phospholipid polar group of the MAPC unit was effectively exposed on the surface as the chain length became longer. Moreover, the hydrophobicity of the surface was increased with the increase in the methylene chain length of the MAPC unit. The number of platelets adhering to the poly(MAPC)-grafted PE surface was reduced from the same point where the P/C ratio became constant.

Animals↗

PubMed1998

Platelet and cell interactions on gold sputter-deposited polymeric surfaces.

Surface treatment as gold sputter-deposited treatment onto various polymeric surfaces has been investigated to improve the cell-, tissue- and blood-compatibility. Surface treated samples were characterized by measurement of contact angle goniometer and electron spectroscopy for chemical analysis (ESCA). The contact angles on the gold-coated polymeric surfaces decreased from 95-65 degrees to around 50 degrees, i.e., increased hydrophilicity due to incorporation of gold thin layer. From the results of ESCA analysis of the modified polymeric surfaces, surface modification by the gold-sputter method was successfully performed. Morphology of the adhered platelets on the gold-coated polymeric surfaces showed lesser activating than control, and the number of adhered platelets surface modified samples decreased with decreasing water contact angle. Fibroblast cell adhesion and growth on the gold-coated polymeric surfaces were more active than those of control. It seems that surface wettability and surface chemistry of gold play important roles for platelet adhesion and cell adhesion, spreading and growth.

3T3 Cells↗

PubMed1997

Interaction of cells on chargeable functional group gradient surfaces.

Functional group gradient surfaces where the surface density of grafted functional groups changes gradually along the sample length were prepared on low density polyethylene (PE) sheets by corona discharge treatment with gradually increasing power and graft copalymerization of acrylic acid (AA), sodium p-styrene sulphonate (NaSS), and N,N-dimethyl aminopropyl acrylamide (DMAPAA). AA and NaSS are negatively chargeable and DMAPAA is positively chargeable in phosphate buffered saline or cell culture medium at pH 7.3-7.4. The functional group gradient surfaces were characterized by the measurement of water contact angle, Fourier transform infrared spectroscopy in the attenuated total reflectance mode, and electron spectroscopy for chemical analysis. All these measurements indicated that the functional groups were grafted on the PE surfaces with gradual increase of their density. The interaction of Chinese hamster ovary cells with the functional group gradient surfaces along the sample length was investigated. The cells that had adhered and grown on the surfaces were counted and observed by scanning electron microscopy. It was observed that a greater quantity of the cells had adhered and grown onto the positions with moderate density of the functional groups. This may be related to the hydrophilicity of the surface. The DMAPAA-grafted surface showed a large amount of cell attachment probably owing to the positive charge character, while the AA-grafted surface, which is negatively charged, showed poor cell attachment, as expected. The NaSS-grafted surface which is also negatively charged showed a large amount of cell attachment. This may be closely associated with the existence of an aromatic ring close to the ionizable group in NaSS. It seems that surface functional groups and their charge character as well as wettability play important roles for cell adhesion, spreading, and growth.

Amines↗

PubMed1997

Cell and platelet adhesions on plasma glow discharge-treated poly(lactide-co-glycolide).

Surface treatment, such as plasma glow discharge treatment, onto poly(lactide-co-glycolide) (PLGA) has been investigated to improve the cell-, tissue- and blood-compatibility. Surface-treated samples were characterized by measurement with a contact angle goniometer and electron spectroscopy for chemical analysis (ESCA). The contact angles on the plasma-treated PLGA surfaces decreased with increasing plasma exposure time from 92 degrees to about 30 degrees, i.e., increased hydrophilicity. From the results of ESCA analysis of plasma-treated films, the incorporation of oxygen molecules into plasma onto the PLGA surface was observed. Cell adhesion and growth on plasma-treated PLGA surfaces were more active than on the control. Furthermore, the maximum adhesion and growth of cells in moderate hydrophilicity were investigated. Morphology of the adhered platelets on the plasma-treated PLGA surface showed less activity than on the control, and the number of adhered platelets on the plasma-exposed PLGA sample decreased with decreasing water contact angle. It seems that surface wettability of PLGA plays an important role in cell adhesion, spreading and growth, and platelet adhesion.

3T3 Cells↗

PubMed1996

Prevention of platelet adhesion on the polysulfone porous catheter by saline solution perfusion, I. In vitro investigation.

A new technique for the prevention of platelet adhesion on the polysulfone (PSf) porous catheter system by means of saline perfusion was investigated. Commercially available ultrafiltration PSf hollow fibers were used as porous catheter tubes. Sixty milliliter of citrated canine blood was circulated for 20 min through the annular catheter flow cell system in vitro. Platelet adhesion onto the PSf porous catheter tube decreased sharply with increased saline solution perfusion rate. From the semilogarithmic relationship between the number of adhered platelets and perfusion rate, the critical perfusion rate for preventing adhesion of platelets could be about 0.3 ml min-1 cm-2. After the surface of PSf porous tubes was modified by non-ionic surfactant coating and air plasma treatment for 20 seconds without saline perfusion, the number of adhered platelets was lower than that of the control tube. These modification methods may be more effective in the prevention of initial platelet adhesion if combined with saline perfusion.

Animals↗