Pouch Of Rats Research Articles

Ectopic bone induction in porous apatite-wollastonite-containing glass ceramic combined with bone morphogenetic protein.

To accelerate the integration of ceramic implants with the surrounding bone and to search for a suitable carrier for bone morphogenetic protein (BMP), we studied ectopic bone induction in porous apatite-wollastonite-containing glass ceramic (A-W GC) combined with partially purified bovine BMP (bBMP) and recombinant Xenopus BMP-4/7 (rxBMP-4/7). Porous A-W GC rods [4 mm in diameter, 5 mm in height, 70% porosity, 200 microns mean pore size, 17.54 +/- 3.82 MPa (mean +/- SD) compressive strength] were used. An apatite coating formed on the surface of porous A-W GC that had been immersed in simulated body fluid at 36.5 degrees C for 7 days. In experiment 1, porous A-W GC rods were combined with either bBMP, collagen, or with both bBMP and collagen. The rods were implanted into subcutaneous pouches in rats and were harvested 4 weeks after implantation. Low-energy radiographic, scanning electron microscopic (SEM), and histological examinations showed ectopic bone formation and within the rods only in the porous A-W GC combined with the bBMP and collagen group. Quantitative analysis also revealed that this group alone showed a significant increase in bone formation. In experiment 2, porous A-W GC rods were combined with rxBMP and collagen, implanted into rats, and harvested as described above. SEM and histological examination showed ectopic bone formation around and within the rods. Because of its relatively high mechanical strength, ease of handling, and good osteoinductivity, porous A-W GC combined with BMP and collagen may be clinically useful in patients with large cancellous bone defects or craniomaxillofacial lesions.

A novel wound dressing with antibiotic delivery system stimulated by microbial infection

The aim of this study was to develop a new wound dressing with controlled release of antibiotics only in the presence of infection. In the first experiment using an infected dorsal pouch of rats, exudate containing proteinases from pouches contaminated with Staphylococcus aureus or Pseudomonas aeruginosa showed significantly higher hydrolytic activity toward Boc-Val-Pro-Arg-MCA than that from noninfected wounds. The authors then developed a new type of wound dressing (AGX), a drug delivery system in which gentamicin is bound to polyvinylalcohol hydrogel through an enzymatically degradable peptide linker containing a -(D)-Phe-Pro-Arg-sequence. To investigate in vitro effectiveness, AGX was incubated with exudate from S. aureus infected or P. aeruginosa infected wounds. Gentamicin was selectively released from AGX in the presence of the exudate from S. aureus infected or P. aeruginosa infected wounds, but was not released in the presence of exudate from noninfected wounds. Next, AGX or the polyvinylalcohol hydrogel that served as control was incubated with S. aureus in the presence of human plasma. After 24 hours, S. aureus concentration was markedly lower in the case with AGX than in that with polyvinylalcohol hydrogel. These results indicate that proteinases from wounds infected with S. aureus or P. aeruginosa cleaved the linker and gentamicin was released.

Analgesic and Anti-inflammatory Activities of the Aqueous Extract of Plantago major L.

This study aimed to validate the pharmacological effects of Plantago major L., a medicinal plant used to decrease pain and inflammation. The aqueous extract (AE) was prepared from the dried ground leaves. Pretreatment with AE (1 g/kg, p.o.) decreased writhing induced by acetic acid in mice, but it did not change the tail flick responses to heat nociceptive stimuli. In rats, AE (1 g/kg, p.o.) reduced the paw edema and pleurisy induced by carrageenin, but it did not alter the paw edema induced by dextran. The effect of AE on the carrageenin inflammatory responses was more intense than that obtained on the ear edema induced by croton oil in mice. In addition, daily treatment with AE (1 g/kg/day during 8 days, p.o.) inhibited the exudative process induced by croton oil injected into the air pouch of rats. The results indicate that the aqueous extract of Plantago major is endowed with effective antiinflammatory and analgesic activities.

Crystallization and preliminary crystallographic data of a neutrophil migration-inducing lectin (KM+) extracted from the seed ofArtocarpus integrifolia

The tetrameric KM+ lectin from the seeds of Artocarpus integrifolia has, when compared to other plant lectins, the singular property of directly inducing neutrophil migration into the peritoneal cavity or into the air pouch of rats. This protein crystals have been grown and they belong to the orthorhombic system with space group C2221. The unit cell parameters are a = 54.4 Å, b = 127.9 Å and c = 99.8 Å. A native diffraction dataset to 2.8 Å was collected and an analysis of the self-rotation function has shown the presence of only one independent non-crystallographic 2-fold axis orthogonal to the crystal b-axis, compatible with a dimer in the asymmetric unit. Proteins 27:157–159 © 1997 Wiley-Liss, Inc.

Crystallization and preliminary crystallographic data of a neutrophil migration-inducing lectin (KM+) extracted from the seed of Artocarpus integrifolia.

The tetrameric KM+ lectin from the seeds of Artocarpus integrifolia has, when compared to other plant lectins, the singular property of directly inducing neutrophil migration into the peritoneal cavity or into the air pouch of rats. This protein crystals have been grown and they belong to the orthorhombic system with space group C222(1). The unit cell parameters are a = 54.4 A, b = 127.9 A and c = 99.8 A. A native diffraction dataset to 2.8 A was collected and an analysis of the self-rotation function has shown the presence of only one independent non-crystallographic 2-fold axis orthogonal to the crystal b-axis, compatible with a dimer in the asymmetric unit.

Ectopic bone formation by composites of BMP and metal implants in rats.

Disc-shaped implants of titanium alloy (Ti-6Al-4V) were treated on one side by corundum-blasting (CB) or by coating with hydroxyapatite (HA) or pure titanium (Ti) using plasma spraying. Half of the implants were additionally coated with purified swine BMP-3. The composites and the uncoated controls were implanted into abdominal wall-muscle pouches of rats. 25 days after implantation, ectopic bone formation could be observed macroscopically and histologically in a high frequency in all 3 groups of BMP-coated implants, whereas the controls were constantly inactive. The volumes of induced bone were similar for BMP-3-coated pure Ti and HA implants, while CB implants were significantly less active. Our findings indicate that the bone formation process is influenced by the chemical composition and by the structure of the implant surface.

Osteogenesis in xenogeneic bone transplantation, using an immunosuppressant. Rabbit-rat experiments.

We investigated osteogenesis and lymphocyte subsets in xenogeneic bone transplantation, using the immunosuppressant FK506 (FK). Iliac bones of rabbits were transplanted as fresh and frozen xenogeneic bone grafts into an intramuscular pouch of rats. FK was injected intramuscularly in half of the rats in a dose of 3 mg/kg/day for 14 days after transplantation. At 2, 4, and 8 weeks, transplanted grafts and the lymphocyte subsets of these rats were examined. In the group not given FK, the grafted bone became necrotic and infiltrated with small round cells around the trabeculae. In the FK group, at 2 and 4 weeks, new bone was formed in the fresh xenografts without infiltration of lymphocytes. At 8 weeks, the new bone became necrotic and lymphocytes were present. The percentage of T cells (CD 5), B cells and the ratio of CD 4 cells/CD 8 cells were smaller in the FK group. Using an immunosuppressant we concluded that xenogeneic bone has an osteogeneic potency.

A role for Bacteroides fragilis neuraminidase in bacterial growth in two model systems.

Two Bacteroides fragilis neuraminidase-deficient mutants were used to study the role of neuraminidase activity in growth of B. fragilis in tissue culture monolayers (CHO cells) and in the in vivo rat granuloma pouch. The nanH structural gene for neuraminidase was cloned from B. fragilis TM4000 and was used to create two isogenic strains with chromosomal disruptions at the nanH gene. B. fragilis VRC404 contains an insertion flanked by disrupted copies of the nanH gene, and B. fragilis VRC426 contains a deletion of a significant portion of nanH coding sequences. The insertion mutant VRC404 is capable of reverting to nanH+. It grew as well as the wild type in CHO monolayers. However, between 48 and 72 h after infection, the bacterial population was enriched with nanH+ bacterial cells (10 to 20%). In the rat pouch 48 h after infection, more than 90% of the population sampled had become nanH+. The deletion mutant VRC426 showed a severe growth defect in the rat pouch model. In addition, VRC426 was efficiently outgrown by the wild type in competition experiments, even when the mutant was present at 10 times the number of wild-type cells at the time of infection. A common characteristic of both model systems is a drastic decrease in the free glucose concentration 16 to 24 h postinfection. We suggest that neuraminidase activity may be required for B. fragilis to grow to maximal levels in the tissue culture and rat pouch systems by making other carbon sources available after glucose levels are reduced.

Induction of neutrophil infiltration by rat chemotactic cytokine (CINC) and its inhibition by dexamethasone in rats

In vivo effects of cytokine-induced neutrophil chemotactic factor (CINC) derived from rats on neutrophil infiltration were investigated using an air-pouch-type inflammation model in rats, and effects of dexamethasone on neutrophil infiltration induced by CINC was also examined in order to gain further insight into the mechanism of antiinflammatory activity of glucocorticoids. Injection of CINC into the air pouch made on the dorsum of rats induced a marked infiltration of neutrophils into the pouch fluid but not mononuclear cells and eosinophils during a 30-min interval after the injection. Maximum effect was induced at a dose of 1.4 micrograms/pouch. Treatment with dexamethasone 3 h before the injection of CINC suppressed the neutrophil infiltration in a dose-dependent manner, but no complete inhibition was observed. CINC injection into the air pouch of rats that had been sacrificed by bleeding in order to minimize neutrophil infiltration from blood stream also stimulated neutrophil infiltration into the pouch fluid when the carcass was incubated at 37 degrees C for 30 min, but the number of infiltrated neutrophils was about 35% of CINC-induced neutrophil infiltration in intact rats. CINC-induced neutrophil infiltration in the carcass, which is supposed to be a reflection of neutrophil migration from extravascular space in subcutaneous tissues to pouch fluid, was not inhibited by dexamethasone treatment. Therefore, the inhibition of neutrophil infiltration by dexamethasone might be due to inhibition of the extravasation of peripheral neutrophils but not due to inhibition of neutrophil chemotaxis from subcutaneous extravascular space to pouch fluid. These findings suggest that clinical effects of steroidal antiinflammatory drugs on neutrophil infiltration in inflammatory disease is partly due to inhibition of neutrophil extravasation induced by preformed neutrophil chemotactic factors in the inflammatory site.

A Nycodenz gradient method for the purification of neutrophils from the peripheral blood of rats

A Nycodenz gradient technique is described which permits the separation of functionally active polymorphonuclear neutrophils (PMN) from the peripheral blood of rats. PMN are obtained at greater than 90% purity and fractionate at a peak density of 1.0919 g/ml. The method is suitable for isolating PMN when the circulating PMN count is low (<20%) as on normal rats and when the count is high (>30%), as a result of inducing inflammation in the subcutaneous air pouch of rats by the intra-pouch injection of peptone. The chemotactic responsiveness of rat PMN was found to be markedly less than that of human PMN when the formyl peptides were used as chemoattractants but not when zymosan-activated serum was the chemoattractant. Blood PMN from normal rats and from peptone-treated rats showed no significant difference in their response to ZAS indicating that priming of their activity by the induction of long term (8 day) inflammation was not a feature of these experiments. However, air pouch-derived PMN displayed a highly significant reduction in activity compared with their isologous blood-PMN. The Nycodenz method offers an alternative to the Percoll separation method for rat blood and will be useful when comparative studies of elicited and non-elicited PMN are required since the latter are obtained in sufficiently high yield and purity for microassays on their function to be performed.

β-Lactamase produced by a highly β-lactam-resistant strain of Barteroides fragilis: an obstacle to the chemotherapy of experimental mixed infections

A strain of Bacteroides fragilis, which produces a metallo-beta-lactamase, was inoculated into pouches on the backs of rats together with a beta-lactamase-negative Escherichia coli highly sensitive to beta-lactam antibiotics. The mixed infection rat pouch model was treated with either flomoxef (susceptible to hydrolysis by the beta-lactamase produced by B. fragilis), or cefmetazole (relatively resistant to hydrolysis). In this model of mixed infection flomoxef showed weak in-vivo activity against E. coli, although showing the same strong activity in a model of single infection with E. coli. On the other hand, cefmetazole showed strong activity against E. coli, even in the model of mixed infection. The concentrations of both drugs in the pouches were decreased in infections with the strain of B. fragilis. There was a greater decrease in the concentration of flomoxef than of cefmetazole. Flomoxef was unstable whereas cefmetazole was relatively stable in the pouch exudates that had been infected with B. fragilis. These experimental data suggest that bacteria that produce a metallo-beta-lactamase decrease the in-vivo efficacy of beta-lactam antibiotics against other co-infecting bacteria. Thus, it is suggested that it is important in the chemotherapy of mixed bacterial infections that include these highly resistant beta-lactamase-producing bacteria to use antibiotics that are stable to hydrolysis by these enzymes.

Cloning and expression of the Bacteroides fragilis TAL2480 neuraminidase gene, nanH, in Escherichia coli

We have cloned the Bacteroides fragilis TAL2480 neuraminidase (NANase) structural gene, nanH, in Escherichia coli. This was accomplished by using the cloning shuttle vector pJST61 and a partial Sau3A library of TAL2480 chromosomal inserts created in E. coli. The library was mobilized into the NANase-deficient B. fragilis TM4000 derivative TC2. NANase-producing colonies were enriched by taking advantage of the inability of TC2, but not the wild-type of NANase+ revertant, to grow in vitro in fluid aspirated from the rat granuloma pouch. Plasmids pJST61-TCN1 and pJST61-TCN3, containing inserts of 9.1 and 4.5 kilobases (kb), respectively, were found in the TC2 derivatives that grew in the rat pouch medium. In B. fragilis, NANase production from the two plasmids was inducible by free N-acetylneuraminic acid or sialic acid-containing substrates, just as in the parental TAL2480 strain. However, when these plasmids were transferred back to E. coli, NANase activity was barely detectable. A 3.5-kb portion of the insert in pJST61-TCN3 was subcloned in pJST61 to give plasmid pJST61-SC3C; NANase was produced from this plasmid both in E. coli and in B. fragilis. In E. coli, NANase expression was under the control of the vector promoter lambda pR and was therefore completely abolished by the presence of a lambda prophage. In B. fragilis, NANase production was inducible by free N-acetylneuraminic acid or sialic acid-containing substrates. By using deletion analysis and Tn1000 mutagenesis, the NANase structural gene and control region that functions in B. fragilis were localized to a 1.5- to 2.0-kb region of the insert. A partial nucleotide sequence of the NANase-deficient Tn1000 insertion mutants allowed us to identify the nanH gene and deduce the amino acid sequence of a portion of the NANase protein. We identified five regions showing great similarity to the Asp boxes, -Ser-X-Asp-X-Gly-X-Thr-Trp-, of other bacterial and viral NANase proteins.

Comparative chemotherapeutic activity of cefpirome and imipenem in experimental infections.

In systemic and local infections, the therapeutic efficacy of cefpirome was compared to that of imipenem and cefotaxime. Murine septicemia induced with methicillin-sensitive and methicillin-resistant Staphylococcus aureus strains responded well to cefpirome and imipenem therapy, the ED50 values ranged from 0.8 to 28.40 mg/kg and 0.5 to 15.58 mg/kg, respectively. The carbapenem also displayed high efficacy against Enterococci and was more potent than cefpirome. Cefotaxime, however, exhibited lower activity or proved to be inactive against these strains. With ED50 values of 0.03 to 31.33 mg/kg, cefpirome was the most active of the three antibiotics in protecting mice challenged with Enterobacteriaceae. The corresponding ED50 values of imipenem and cefotaxime ranged from 0.72 to 70.95 mg/kg and 0.06 to 66.30 mg/kg, respectively. Despite distinctly lower in vitro activity against the infecting organism, cefpirome showed efficacy similar to imipenem in the treatment of subcutaneous S. aureus abscesses in mice. It was more effective than imipenem and cefotaxime against experimental Klebsiella pneumonia in mice and the Escherichia coli infected granuloma pouch in rats.

Anti-inflammatory and Antipyretic Activity of Euphorbia peplis L

AbstractThe ethanolic extract of Euphorbia peplis L. has been screened for anti-inflammatory, analgesic and antipyretic activities in suitable experimental models. It produces significant inhibition of carrageenan-induced paw oedema and cotton pellet-induced granuloma pouch in rats and a significant reduction of yeast-induced hyperpyrexia in mice. It failed to produce any analgesic activity on the hot plate reaction time test. On hematological parameters contradictory observations were recorded as it showed a proaggregatory effect on platelets and reduced fibrinogen level without affecting prothrombin time. Among the phytoconstituents detected in this plant, flavonoids may be responsible for the observed anti-inflammatory effect of the ethanolic extract.

Adjuvant polyarthritis and the pseudosynovitis.

The cavity lining of a six day old subcutaneous air pouch in rats resembles normal synovium in morphology and responds in a comparable manner during development of polyarthritis. The ability of newly formed pouches (one and two days old) to respond similarly to polyarthritogen was examined here. Positive macro and microscopic changes similar to those seen in the six day old pouch were observed in rats previously inoculated with adjuvant. The intensity of some of these changes varied between pouches. The results of this study indicate that the adjuvant induced air pouch reaction does not depend on the presence of synovial-like lining for its production.

Pharmacological investigations of Hibiscus micranthus Linn., a febrifuge used in Saudi Arabian folk medicine

AbstractThe ethanolic extract of Hibiscus micranthus Linn. has been studied for its pharmacological activities. It has been found to produce significant inhibition of carrageenan induced paw oedema and cotton pellet induced granuloma pouch in rats and significant reduction in yeast induced hyperpyrexia in mice. It failed to produce any analgesic activity on the hot plate reaction time test in mice. On the hematological and biochemical parameters it produced a significant prolongation of prothrombin time, a decrease in the fibrinogen content and marked hyperglycemia in rats. It also produced significant neuromuscular blocking activity on both the models used in this study. Apart from these, it exhibited slight inotropic and chronotropic effects on isolated rabbit heart and a mild CNS stimulant effect. Further studies on the isolation of its active phytoconstituents and their pharmacological actions are being undertaken.

Mutagenicity of 2-amino-3-methylimidazo[4,5-ƒ]quinoline (IQ) in the granuloma pouch assay

The rat granuloma pouch assay was used to assess the in vivo mutagenic potential of 2-amino-3-methylimidazo[4,5-ƒ]quinoline (IQ), a heterocyclic aromatic amine which is formed during the frying of meat and broiling of fish. The assay was performed with and without pre-induction by Aroclor. In the initial experiment IQ was injected directly into the pouch of non-induced rats. A 10-fold increase in mutation frequencies was obtained with the 2.0 mg/pouch dose of IQ with uninduced cell populations. In a second study IQ was injected intraperitoneally and into the pouch of rats that had been pre-induced with Aroclor. The dose of IQ administered varied from 0.1 to 2.0 mg/pouch. A 10-fold increase in mutation frequencies was obtained with the 2.0 mg/pouch dose of IQ with uninduced cell populations. Aroclor treatment produced no significant increase in mutation frequencies over uninduced animals. Its mutagenic effect is about 10-fold weaker than that of benzo[ a]pyrene or N-methyl- N′-nitro- N-nitrosoguanidine (MNNG).

Involvement of glucocorticoid in insulin-induced angiogenesis of adjuvant pouch granuloma in diabetic mice.

The effects of insulin and glucocorticoid on granuloma formation and angiogenesis were studied using an adjuvant pouch in mice and a carrageenin pouch in rats. Carrageenin-induced granuloma formation was suppressed in the diabetic state induced by alloxan in rats. The suppression was restored by adrenalectomy. Corticosterone counteracted the restoration, whereas epinephrine did not, suggesting that the effects of adrenalectomy are due to the lack of adrenocortical hormones rather than epinephrine. The blood corticosterone levels in alloxan mice increased, following the increase of glucose level, to more than 20 micrograms/dl after 4 weeks. The increase of corticosterone levels disappeared after adrenalectomy. In the mouse adjuvant pouch, corticosterone dose-dependently decreased granuloma formation and angiogenesis. The values of these two parameters obtained with a dose of 20 micrograms/pouch corticosterone agreed with those of the serum levels in diabetic mice. Insulin dose-dependently reversed the suppressed angiogenesis of 20 micrograms corticosterone-treated mice and the dose-response curve approximated the curve of alloxan-treated mice. The effects of diabetes were concluded to involve insulin deficiency as well as glucocorticoid enhancement and the counteraction between the two may control granuloma formation and angiogenesis.

Effect of the basal diencephalon on the development of Rathke's pouch in rats: A study in combined organ cultures

Rathke's pouch of 12.5-day-old fetal rats was cultured alone or with various fetal organs. After 10 days of culture with the anterior or posterior wall of the basal diencephalon, the size of the pouch was about eight times larger than that in single culture or in combined culture with the telencephalon or liver anlage. There was no chronological difference in the appearance of immunoreactive ACTH and prolactin cells between single and combined cultures. These results suggest that fetal diencephalon promotes the growth of Rathke's pouch but has no potency to promote differentiation of the pituitary cells.

Anti-inflammatory activity of a proteolytic enzyme, Prozime-10.

Prozime-10 (P-10), a proteolytic enzyme extracted from cultured broth of Aspergillus melleus, was injected intravenously into rats in dosages of 1--10 mg/kg and was found to alleviate carrageenin edema. Thermal denaturation diminished not only the proteolytic activity but also the antiedematous effect. Carrageenin-induced granuloma pouches in rats were reduced by P-10 in a dose-dependent manner (1--5 mg/kg). With P=10 (5 mg/kg i.v.), adjuvant arthritis was inhibited by 62% in the primary phase and by 57% in the secondary phase. Serum concentration of immunoprecipitable P-10 reached 100 mg/l within 30 min after the intraduodenal administration of P-10 (200 mg/kg) and was concomitant with a 3-fold increase in the blood esterolytic activity. When P-10 was injected (5 mg/kg i.v.) into rats, the plasma corticosterone level increased to a maximum of 4 times the preinjection level. These observations indicate that: (a) P-10 can be absorbed from the gut in sufficient quantities to bring about anti-inflammatory activity, and (b) the anti-inflammatory activity of P-10 can be related, in part, to the ability of P-10 to affect the release of glucocorticoids.