Umeko Kawaharada

In vivo controlled release of a luteinizing hormone-releasing hormone agonist from poly(dl-lactic acid) formulations of varying degradation pattern

Abstract Biodegradable formulations with a desired lag time were prepared from blends of low molecular weight poly( dl -lactic acid) (low MW-PLA, number-average molecular weight: M n = 1400 , parabolic-type degradation pattern, 100% in vivo degradation at 5th week of implantation) and high molecular weight poly( dl -lactide) (high MW-PLA, M n = 11 500 , S-type degradation pattern with a lag time of 10 weeks). A luteinizing hormone-releasing hormone agonist (LH-RH agonist), des-Gly10-[Leu6]LH-RH ethylamide monoacetate, was incorporated into the small cylinders of PLA blends. The initial burst of drug release from cylindrical formulation, which was implanted subcutaneously in the back of male rats, could be controlled by adjusting the amount of high MW-PLA in the blend and, as a result, it was found by measuring the pharmacological influence on rat prostate and serum drug levels that the best efficacy and the most constant release over a long period are obtained with a 25/75% low MW-PLA/high MW-PLA blend.

Localization of metallothionein in aged human brain.

The localization of metallothionein (MT), a small molecular weight heavy metal binding protein in aged human brain, was investigated by immunohistochemical techniques. The amount of MT and heavy metals (Zn, Cu) were also assayed by radioimmunoassay and atomic absorption spectrophotometry, respectively. Immunohistochemically, MT was found in the pia mater, ependymal cells, protoplasmic astrocytes nand glial processes neuropil of the gray matter and fibrous astrocytes of the white matter of the telencephalon, whereas oligodendroglia and microglia did not show any positive immunostaining for MT. Cytoplasm, glial processes and some nuclei of protoplasmic and fibrous astrocytes showed strong MT immunostaining. Vascular feet and adventitia were also positive for MT immunostaining. Moreover, the pia mater, astrocytes and ependyma in the diencephalon, mesencephalon, pons, medulla oblongata and spinal cord showed the same positive immunostaining for MT as the telencephalon. In the cerebellum, Bergmanns glia, protoplasmic astrocytes of the granular layer and fibrous astrocytes of the white matter showed strongly positive immunostaining for MT.

In vivo release of cisplatin from a needle-type copolymer formulation implanted in rat kidney

Cisplatin, cis-dichlorodiamine platinum (II), was incorporated in a needle-type copolymer formulation (0.8 mm diameter, 6 mm long) by radiation-induced polymerization. The copolymer used was copoly(diethylene glycol dimethacrylate/polyethylene glycol #600 dimethacrylate, 80/20 vol%). This copolymer, containing 6 mg of cisplatin, was implanted into the kidney of adult male Wistar rats (420 +/- 20 g). A total of 70 d was required for 100% release of cisplatin in vivo. The kidney tissue surrounding the formulation was strongly necrotized by the action of cisplatin. Two layers of necrosis could be distinguished: necrotic tissue surrounding the formulation and necrobiotic tissue surrounding the necrotic tissue. The amount of necrotic tissue changed markedly over time, but no change was apparent in the amount of necrobiotic tissue. The maximal amounts of necrotized tissue were observed 14 d after implantation: 3100 microns and 600 microns thick for the necrotic and necrobiotic tissues, respectively.

Studies of the slow releasing of testosterone from radiation-polymerized testicular prostheses implanted subcutaneously in the back of castrated rabbits.

A controlled release testicular prosthesis containing testosterone, which was previously dissolved in 2-hydroxyethyl methacrylate (HEMA) at a temperature of 80 degrees C, was prepared by radiation-induced polymerization in the supercooled state at a low temperature. The daily dose of testosterone released in vitro from the poly(HEMA) testicular prosthesis was kept constant at a rate of 5.5 +/- 1.5 mg/d throughout an experimental period of 900 d. In the in vivo experiments, the poly(HEMA) testicular prosthesis was implanted subcutaneously in the back of castrated rabbits over a maximum period of 11 mnth. The cumulative amounts of testosterone released in vitro and in vivo from the poly(HEMA) testicular prosthesis for a period of 11 mnth were found to be 2.1 g (30.0 wt% of initial drug) and 0.9 g (12.8 wt% of initial drug), respectively. The serum testosterone level in castrated rabbits with a poly(HEMA) testicular prosthesis rapidly decreased for periods up to 2 mnth (after increasing during the first 2 wk), then showed a moderate decrease for a few months, and finally held constant at a level of 10 ng/ml throughout the experimental period. It was concluded that a slight amount of testosterone is continuously released in vivo from the radiation-polymerized poly(HEMA) testicular prosthesis over a long period analogous with that in vitro.

Immunohistochemical localization of metallothionein in plant tissues

The localization of metallothionein ( MT ) in the seeds and roots of soybean was investigated by immunohistochemistry. The germinating seeds at 2 hr, 1, 2, 3, 4 and 6 d including 1-mo root tips of soybean ( c.v. Toyosuzu ) with and without heavy metals ( Cu 400 μg Lor Zn 3 μg ml−1) treatment were used to demonstrate the localization of MT by the indirect immunoperoxidase technique using polyclonal rabbit antirat MT conjugated to ascaris as a primary antibody. Metallothionein was localized in the proliferating regions such as the embryo in seeds, and root and shoot apices of both the control and heavy metals-treated plants. The intensity of MT staining in the proliferating regions generally increased as the soybean seeds germinate. Starting at about 1 day after germination, MT was found in the veins and vascular bundles suggesting its translocation to other organs. Similar observation hold true in the case of plants treated with heavy metals. This means that heavy metals treatment had no effect on MT localization. However, the heavy metals-treated plants showed higher concentration of MT over the control with respect to the growth stage of soybean seeds. These indicate that MT found in soybean plays a physiological role in heavy metal transport, detoxification and cell division in a similar manner to mammalian MT.

LIGHT AND SCANNING ELECTRON MICROSCOPIC AND IMMUNOHISTOCHEMICAL STUDIES ON PERMEABILITY OF HYPERTENSIVE RAT MESENTERIC ARTERIES

Experimental hypertensive rats were intravenously injected with carbon and iron as tracers, and their mesenteric arteries exhibiting hypertensive arterial lesions were observed by light and scanning electron microscopy and immunohistochemistry. Early arterial lesions showing entense medial damages, deposition of fibrinoid substance consisting of fibrin in the intima and/or media, and granulation tissue in the adventitia were characterized by marked insudation of intravenously injected tracers. Scanning electron microscopy demonstrated numerous leukocytes and platelets adhering to endothelial surface, opened endothelial cell junctions, and desquamation of these cells. Immunohistochemistry revealed laminin and low stainability of fibronectin in the subendothelium. Advanced lesions showed deposition of a large amount of fibrinoid substance and no insudation of tracers in the intima, but scanning electron microscopy manifested opening of endothelial cell junctions, desquamation of endothelial cells, and adherence of leukocytes and platelets. Immunohistochemistry revealed fibronectin in the intima and laminin just beneath the endothelium. In the healed lesions disclosing fibrocellular intimal thickening, there was no insudation of tracers. Scanning electron microscopy showed opened endothelial cell junctions, endothelial cell defects, and adherence of leukocytes and platelets. There were fibronectin in the intima and laminin beneath the endothelium. It was suggested that the opening of endothelial ceils junctions and desquamation of endothelial cells would be necessary for the arterial increased permeability in hypertensive rats, and that fibrin‐fibronectin complex, fibronectin‐acid mucopolysaccharide complex, and basement membrane would together inhibit the increased permeability in the mesenteric arteries of hypertensive rats in spite of endothelial cell injuries and their defects.

BASAL CELL ADENOMA WITH PARALLEL TUBULES IN STROMAL CELLS OF THE PAROTID GLAND

A case of basal cell adenoma in the right parotid region of a 51 years old male was reported. The tumor measured 2.5 cm x 3 cm, was spherical and covered with a fibrous capsule. Histologically, it was a tubular monomorphic adenoma with scant edematous interstitial tissue. The stromal cells stained positively by the PAP method using anti‐S‐100 protein serum. Electron microscopically, the tumor cells forming tubules had many microvilli at the luminal surface, many filaments in the cytoplasm and well developed des‐mosomes in the intercellular junctions. Ordinary intracellular organelles of the tumor cells were small in number, and their nuclei were oval with shallow indentation. In the dilated rough endoplasmic reticulum of the stromal cells, many straight parallel tubules were found. The tubules measured from 15 nm to 25 nm thick and 3.5 γm long in the longitudial sections and from 25 nm to 30 nm in diameter with electron lucent core and poor coat in the cross sections. Other cell organelles of the stromal cells were small in number, and filaments and dense attachments were found in the ectoplasm. Around the stromal cells there was a discontinous basement membrane. ACTA PATHOL. JPN. 34 : 1449–1458. 1984.

ROLE OF LYSOSOMES IN CORONARY ARTERY LESIONS OF HYPERTENSIVE RATS

Electron microscopically the role of acid phosphatase (acid P) positive lysosomes in the patho?enesis of intramyocardial coronary artery lesions of hypertensive rats with bilaterally constricted renal arteries was studied. At 3 postoperative weeks, acid P positive lysosomes were increased in endothelial cells of the coronary arteries, but at 5 weeks and thereafter, they were decreased in number. In the intima, intimal smooth muscle cells with acid P positive lysosomes appeared at 3 postoperative weeks, but their number remained small as late as 9 postoperative weeks. The internal elastic lamina was fra?mented into amorphous masses from 3 postoperative weeks, at 5 weeks and thereafter the fra?mentation and disruption became severer, and at 9 weeks the lamina disappeared because of marked disruption. At 3 postoperative weeks, acid P positive extracellular lysosomes were observed in the ?aps of the fra?mented internal elastic lamina. At 5 weeks and thereafter, extracellular lysosomes and attenuated processes of medial smooth muscle cells with lysosomes were seen extending through the ?aps of the fra?mented internal elastic lamina. Necrosis of these extendin? cell processes and lysosomes remainin? after the necrosis were observed. The findin?s su??ested that the fra?mentation and lytic chan?e of the internal elastic lamina observed in hypertensive rat intramyocardial coronary arteries might be induced by extracellular lysosomes dischar?ed not only from endothelial cells and intimal smooth muscle cells but also from the extendin? processes of medial smooth muscle cells into the gaps of the internal elastic lamina. ACTA PATHOL. JPN. 36: 1529‐1536, 1986.

ROLE OF LYSOSOMES IN MESENTERIC ARTERIAL LESIONS OF RENAL HYPERTENSIVE RATS

The acid phosphatase activities of arterial cells in the mesenteric arteries of renal hypertensive rats were investigated by both light and electron microscopy. Light microscopically, strongly positive acid phosphatase reactions were confirmed in endothelial cells, intimal cells, medial cells and adventitial cells of the mesenteric arteries, together with considerable deposition of fibrinoid substance in the intima and media in contrast to the appearance in control rats. Electron microscopically, lysosomes with acid phosphatase‐positive reaction products were increased in number in endothelial cells, intimal smooth muscle cells, medial smooth muscle cells and adventitial neutrophils or macrophages. The lysosomes in intimal smooth muscle cells and those which were extracellularly discharged were responsible for lysis of the fibrinoid substance. In the media, acid phosphatase‐positive lysosomes of medial cells and extracellularly discharged matrix lysosomes with acid phosphatase‐positive reactions were also involved in the hydrolysis of necrotic substances and extracellular matrix. These acid phosphatase‐positive reactions were diminished both light and electron microscopically in endothelial cells, intimal cells, medial muscle cells and adventitial cells in the regions of healing arteries where fibrinoid substance had been degradated and the intima showed cellulofibrous thickening. The possible role of this acid phosphatase activation for the clearance of cell debris as well as exudative substances in the healing of damaged arterial tissue was discussed.