실험적 Endotoxin Shock에 있어서의 췌장외분비 및 내분비세포의 초미형태학적 연구

권건영,정재홍,손태중 계명대학교 醫科大學 病理學敎室同門會 1987 樂山 鄭在泓 敎授 頌喜 紀念論文集 Vol.S No.-

저자는 endotoxin 으로 인한 췌장의 외분비 및 내분비세포의 상해기전의 일단을 알아보기 위하여 Sprague-Dawley 종 흰쥐에 endotoxin 을 체중 1 kg 당 7.6 mg을 복강내에 투여한 후 30분, 1, 2, 4, 6, 8시간에 췌장 장을 절취하여 광학현마경, 투과전자현미경 및 주사전자현미경으로 검색하고 아울러 lysosome 내 의 으CP으se 의활성을 전자현미경적 효소세포화학적 방법으로 관찰하였다. 그 성적을 요약하면 다음과 같다. 광학현미경으로는 외분비부 및 내분비부에 1시간후부터 경한 간질의 부종과 충혈이 일어나기 시작하여 2시간째에는 이들 변화가 좀더 심하여졌고 또 여기에다 더하여 세포질의 공포화 및 혈관주위의 부종이 출현하였고, 4시간후에는 2시간후의 변화와 더불어 소동맥중 막의 공포가 출현하였고, 6시간 및 8시간째에는 상기한 모든 변화들이 더 심하여지고 또 여기에 내 ·외분비세포의 염색성의 저하가 일어났다. 투과전자현미경으로는 외분비세포에 있어서는 endotoxin 투여 후 30분에 있어서는 경한 pre-lysosome 의 증가만을, 1 시간에는 prelysosome 이 더욱 증가되었고, 여기에 더하여 secondary lysosome 의 출현,mitochondri의 종창 및 cristae 의 감소, RER 의 충상배열의 혼란, 수포성 성분의 출현, Golgi 장치에서는 공포성분의 증가, 그러고 분비과립에 있어서는 과립의 숫적 감소 및 그 내용물의 전자밀도가 감소하는 등 주로 변성성변화들이 출현하였는데, 이들 소견은 시간이 경과할수록 더 심하여지는 경향이었다. 내분비 세포에 있어서의 변화는 외분비세포에 있어서의 소견과질적으로 거의 같았으나 출현시간이 늦어서1시간내지 2시간째부터 나타났으며 그 정도는 경한 편이었다. 모세 혈관에 있어서는 endotoxin 투여 후 30분부터 혈관주위의 부종, 내강의 확장, 충혈, 내피세포에는 세포질의 부종, pinocytotic vesicle 의 증가등이 일어나 기시작하여 1 시간 이후에는 이들 변화가 더욱 심하여 져서 내파세포핵의 농축, heterochromatin 의 증가,세포간격의 확장, 내피세포 세포질의 강내로의 돌출,pinocytotic vesicle 의 증가등이 매우 현저하여 졌고 2시간 이후에는 이들 현상이 더욱 심해졌고 여기에다 내피세포의 파괴와 미소혈전의 형성등이 관찰되었다. 전자현미경적 효소세포화학적 관찰에서는 pre-lysosome 과 post.lysosome 에 서 ACPase 활성이 인지되지 않았으나, primary lysosome 에서는 균질한 활성이 , secondary lysosome 에서는 부분적인 활성이 나타났다.주사전자현미경으로는 외분비 및 내분비세포에서는 특이한이상을 관찰할 수 없었고, 혈관 특히 모세혈관, 세동맥 및 정맥에는 내피세포의 종창, 수포형성, 배열의 불규칙 , 내강의 협착, 충혈 및 미소혈전등이 endo-toxin 투여후1 시간 부터 관찰되었으며, 6시간후 부터는 내피세포의 단열이 관찰되었다. 이상의 성적 으로 보아 endotoxin 투여로 인한 췌장 상해 발생기전은 endotoxin을 실험 동물에 투여하면,이는 혈류에 들어가서 헐관내피세포에 먼저 상해를 주고 이로 인하여 혈관내의 액체성분이 혈관밖으로 유출 되어 그 주위조직에 부종을 일으키고 나아가서는 혈전을 형성하여 외분비 및 내분비 세포로 가는 혈류의 감소내지 정지를 일으켜 그 결과 외분비 및 내분비세포내의 각 구조물에 퇴행성변화가 일어나며 이 퇴행성 물질을 소화, 처리하기 위하여 먼저 pre-lysosome, 이 어서 primary lysosome 및 secondary lysosome 이 , 그리고 마지막으로 post-lysosome 이 증가하는 것 이 라믿어진다. 그러나 이때 혈류에 들어간 endotoxin 이직접 외분비 및 내분비 세포에 도달하여 상해할 가능성도 있다고 사료된다.

血中 Ammonia 微量定量에 있어서 pH 및 Alkali 媒劑가 測定値에 미치는 影響에 對한 硏究

鄭在泓 계명대학교 醫科大學 病理學敎室同門會 1987 樂山 鄭在泓 敎授 頌喜 紀念論文集 Vol.S No.-

An attempt was made to determine the origin of artifactual blood ammonia formed in the shed blood; Experimentation, with blood specimens from more than 400 healthy individuals, was carried out over a period of four and a half years, utilizing two popular microdiffusion blood ammonia methods, those of Seligson and Conway. The actions of several alkali [pstassium carbonate, carbonate-bicarbonate mixture and borate-sodium hydroxide (with a pH of 9 to 11.4)] were compared by placing them in the diffusion apparatus with specimens of blood. It was found that the ammonia concentration tended to be higher with increasing pH and vice versa lowering with decreasing pH. While occasional extremely implausible results were obtained in experiments using the potassium carbonate and carbonate-bicarbonate mixture, on the other hand, no such aberrations were observed with borate-sodium hydroxide. Within the pH range, from 9 to 10.1, the yield of ammonia when plotted against the pH of the blood-alkali mixture showed a straight line. Increasing beyond 10, resulted in an asymptomatic character of diffusion curve. For these reasons, borate-sodium hydroxide reagent which results in a pH of 10.1 in the presence of blood, is selected as the choice of weak alkali media for the determination of true blood ammonia. The author has to abandon his earlier thought that aberrations in ammonia analyses can be largely attributed to faulty technique in collecting and handling blood. Careful control of these techniques resulted in some lowering of the ammonia concentration, but failed to prevent the more serious aberrations encountered. In search for clarification of the origin of extra ammonia, studies were carried out by experimenting with glutamine, both in aqueous solutions and following mixture with blood. The results suggest that non-protein glutamine is an unlikely source of extra ammonia. The only other plausible source of the extra ammonia liberated from blood, therefore, seemed to be the blood proteins, Accordingly, the action of various alkaline reagents on plasma albumin, globulin and on whole blood was tested, after prolonged dialysis to remove preformed ammonia. Ammonia was released from each of the protein solutions used. However, the action of borate in this release was less pronounced than that of the carbonate-bicarbonate mixture. This latter mixture, in turn, liberated substantially less ammonia than did potassium carbonate. Therefore, it is concluded that the origin of artifactual ammonia may be traced to the decomposition of protein by the action of alkali. The author has revised the method of measuring blood ammonia in such a way as to avoid spontaneous production of ammonia from blood proteins. The technique involves the use of sodium borate buffer at a pH of 10.8±0.2, which, when mixed with blood, results in a pH of 10.1 The adequacy of this technic is well proven by the satisfactory results obtained in a large number of recovery studies. The technique described, applied to venous specimens that were collected in EDTA tubes and promptly analyzed, was used to study a group of 16 healthy persons, 20 to 25, male, with no history of previous liver involvement. The mean blood ammonia was 0.42 μg./ml. expressed as N. The lowest value was 0.21, the highest, 0.61, with standard deviation of ± 0.124.

Sodium iodate에 의한 실험적 망막외층 손상시의 망막전위도 및 미세구조 변화

安炳憲,尹東浩 계명대학교 醫科大學 病理學敎室同門會 1987 樂山 鄭在泓 敎授 頌喜 紀念論文集 Vol.S No.-

The purpose of the present study was to investigate certain relationship between ultrastructural and electrophysiological changes in the retinal degeneration induced by sodium iodate. 1n the previous reports of other authors, functional changes of the degenerated retina.always preceded the structural changes even in the ultrastructure. The author investigated the sequence of intracellular ultrastructural changes in the earlier stage of retinal degeneration comparing with the electroretinographic changes. Twenty seven rabbits were given an intravenous injection of 2 % sodium iodate. A single does of 50 mg/kg body weight produced relatively constant patterns in electroretinographic and ultrastructural changes in 14 rabbits. The amplitudes of the 3 component waves of electroretinogram increased transient 15 minutes after sodium iodate injection. One and half hours later, b and a waves decreased. The electroretinogram disappeared completely 24 hours later and did not recover with time passing up to 3 weeks. The first ultrastructural changes were observed in the pigment epithelium only 15 minutes after sodium iodate injection. The pigment epithelium showed mild swelling of mitochondria and dilatation of smooth surfaced endoplasmic reticulum. The basal infoldings and apical villi were still preserved at thattime. The space between the cristae was wide but their outlines were visible. The cellular changes progressed and ended in necrosis about three days later. By the time photoreceptor cells changed also and left severely deformed inner segments over the degenerated pigment epithelium and Bruch’s membrane with complete loss of outer segments. A week later some repairing process occured in the pigment epithelium. A lot of modified pigmented cells lay on the Bruch’s membraneand appeared quite different from the normal ones. The cytoplasm of mueller cell extended through a gap in the external limiting membrand. They participated in supporting the degenerated photoreceptor inner segments.

Aminopyrine 및 Heparin이 全身性 Shwartzman 反應에 미치는 影響에 關한 實驗的 硏究

金貞淑 계명대학교 醫科大學 病理學敎室同門會 1987 樂山 鄭在泓 敎授 頌喜 紀念論文集 Vol.S No.-

The generalized Shwartzman reaction is produced experimentally by intravenous administratipn of two successive doses of endotoxin derived from Gramnegative micro-organisms, spaced approximately 24 hours apart, in susceptible animals, such as rabbits and rats. The initial or preparatory injection of endotoxin given to the animals in' producing the generalized Shwartzman reaction can be substituted by administration of reticuloendothelial blocking agents, such as trypan blue, thorotrast, cortisone, and ethyl stearate. Such blocking is also observed in pregnancy. The fundamental pathological change of this interesting but pathogenetically complicated generalized Shwartzman reaction is formation of glomerular fibrin thrombi and renal cortical necrosis. There have been many studies on the prevention of Shwartzman reaction during the past two decades and a variety of preventive agents .have been introduced; namely, ' nitrogen mustard, aminopyrine, dibenamine, streptokinanse, sodium warfarin, hirudin, and heparin. Among the variable results, aminopyrine and heparin were found to be effective to prevent the localized and classical generalized Shwartzman reactioD. There has been no. report OD the inhibitory effect of heparin on the eneralized Shwartzman reaction induced in pregnant rabbits or in the human couDterpart. The latter has been reported recently in abortions, predominantly of a criminal nature, with a fatal outcome resulting from the generalized Shwartzman reaction induced by Gram-negative organisms. This investigation is an attempt to evaluate the inhibitory effect of aminopyrine on the generalized Shwartzman reaction induced by a classical two-dose intravenous injection of E. coli endotoxin into non-pregnant rabbits and that of heparin on the generalized Shwartzman reaction induced by a single intravenous injection of E. coli endotoxin iD pregDant rabbits_ Fifty-two albino rabbits were used in this study, divided into 3 groups. Group 1. Twenty non-pregnant rabbits of both sexes were used to determine the endotoxin .doses necessary to induce classical generalized Shwartzman reaction by giving two successive intravenous injections, 24 hours apart. Four out of 20 animals were designated to decide the optimal doses of E. coli endotoxin that can produce the expected generalized Shwartzman reaction Group 2. Twelve non-pregnant rabbits of both sexes .were used to evaluate an inhibitory .effect of aminopyrine on generalized Shwartzman reaction The animals were subdivided into an aminopyrine group and a control group. First, the animals of aminopyrine group, 10.0, 0.3 and O. 25 mg. of aminopyrine were administered intravenously 40 minutes before provocative injection of endotoxin. Second, the control group animals were given a two-dose intravenous injection of endotoxin in the same manner that is described in group. 1 Group 3. To observe the preventive effect of heparin against an incipient generalized Shwartzman reaction, 20 pregnant rabbits were used. They were subdivided into a heparin group and a control group. The animals of the control group were given a single intravenous injection of E. coli endotoxin and those of the heparin group were treated as follows: ⓐ An intravenous 'injection of heparin simultaneously with the E. coli endotoxin injection. ⓑ An intravenous injection of heparin 2 hours later. ⓒ An intravenous injection of heparin 4 hours later. The results were as follows: Group 1. The optimal doses of endotoxin that can induce generalized Shwartzman reaction were varied from 2.5 to 4.2 mg_ Thirteen out of 16 animals (81%) revealed fibrin thrombi in the glomerular capillaries and of these, 5 animals (31%) bilateral cortical necrosis occurred. Fibrin thrombi were also observed in the pulmonary capiIlaries in 12 (75 %) of these animals. Group 2. Aminopyrine failed to demonstrate an inhibitory effect On the generalized Shwartzman reaction, contrary to previous reports that it is capable of preventing Shwartzman reaction of a localized form. Group 3. Generalized Snwartzman reaction was induced with E. coli endotoxin of 3.5, 3.6 and 5.4mg. given as a single provocative intravenous injection into the pregnant rabbits in the control group. The counterpart, heparin treated animals, failed to show any evidence of morphological change that can be construed to be a phenomenon of a generalized Shwartzman reaction. The fall of fibrinogen level in this control group with generalized Shwartzman reaction seems to be in keeping with the concept that intravenous coagulation plays an important role in the pathogenesis of this reaction It can be concluded, therefore: 1. Optimal doeses of E. coli endotoxin which induce generalized Shwartzman reaction in non-pregnant rabbits can be varied from 2.5 to 4.2 mg. 2. Aminopyrine which is known to prevent local Shwartzman reaction failed to inhibit generalized Shwartzman reaction in non-pregnant rabbits. 3. Generalized Shwartzman reaction was produced in pregnant rabbits of the control group by a single intravenous injection of E. coli endotoxin and the reaction that was produced in the above was prevented when the animals were treated with heparin. A fall in the fibrinogen level of the pregnant rabbits with generalized Shwartzman reaction in the control group appears to be well correlated with the intravenous coagulatlon phenomenon that took place in the above reaction

Ultrastructural Morphogenesis of Mitochondria in Alcoholic Liver

Chang Eun Sook 계명대학교 醫科大學 病理學敎室同門會 1987 樂山 鄭在泓 敎授 頌喜 紀念論文集 Vol.S No.-

Electron microscopic studies of 53 diagnostic liver biopsies from 23 patients were done. Ultrastructural study revealed most striking alteration to be in the mitochondria such as pleomorphism in shape and size, especially giant mitochondria with disorganization, crystalline inclusions and calcium-like electron dense bodies. These ultrastructural changes might have been induced by specific bichemical abnormality in alcohol bearing condition. Diagnostic use of electron microscopy will be useful in detecting alcoholic injury in a1coholic patient who fails to demonstrate any discernible laboratory and light microscopic abnormalities. ACTA PATHOL. JPN. 37: 213-224, 1987.

실험적 안내염이 망막전이도 및 망막미세구조에 미치는 영향

鄭欽 계명대학교 醫科大學 病理學敎室同門會 1987 樂山 鄭在泓 敎授 頌喜 紀念論文集 Vol.S No.-

The endophthalmitis as a complication after intraocular surgery usually takes a tragic course, and its early diagnosis and proper management are urgently mandatory to preserve the vision and the eyeball. Furthermore, functional suppression of the retina can be anticipated in the early stage of endophthalmitis by the toxic effect of the substance produced by the growing organisms. Bacterial endophthalmitis was experimentally induced in the rabbit eyes by inoculating Staphylococcus aureus (1. 25 X 103 organism/0.05ml) intravitreally, and the sequential changes of electroretinogram (ERG) and retinal ultrastructure were correlated. The clinical signs of endophthalmitis firstly appeared 15 hours after inoculation with increasing severity thereafter. However, the amplitude of ERG, both a-and b-waves, began depression 3 hours after inoculation, reaching down to 45-56% of level of preoperative amplitude at 18 hours and totally flat 30 hours later. Ultrastructurally, the retinal pigment epithelium (RPE) was the only site of involvement by 18 hours, being manifested from 7 hours group by dilatation of smooth endoplasmic reticulum and mitochondrial swelling as well as detachment of ribosomal granules from rough endoplasmic reticulum, formation of myelin figures and fat droplets. Subsequent injuries beyond those in the RPE were extension of similiar changes to the inner segment of photoreceptor and to the outer nuclear layer 24 hours later. Based on the earlier demonstration of abnormality in the ERG rather than clinical sign or ultrastructural changes, especially with falling of its amplitude down to 50% or less of preoperative level after 18 hours, and reversibility of ultrastructural alterations of retina by the same period of time, it is assumed that an ear1y diagnosis by using ERG and prompt application of management before the amplitude .of ERG falls below 50% will give a beneficial effect on the bacterial endophthalmitis.

骨髓腔內 釘揷入이 骨折治療에 미치는 影響에 관한 實驗的 硏究

金益東 계명대학교 醫科大學 病理學敎室同門會 1987 樂山 鄭在泓 敎授 頌喜 紀念論文集 Vol.S No.-

Fifty-eight rabbits were subjected, to fracture and studied. Linear or segmental fractures at the junction of the middle and distal thirds of both . radii of these animals were made. In all experimental animals a Kirschner wire was inserted intramedullary from the distal end of the right radius. The left radius was not fixed with any internal device and was used as the control side. Animals were sacrificed and studies made at 1 week, 10 days, 2, 3, 4, 5, 8, 12, and 16 weeks. Radiography, microangiography, biochemical, and histological techniques were used. The results were as follows; 1) Radiography: The side with intramedullary fixation formed the periosteal callus earlier than the control side. Though callus formation appeared earlier in segmental fracture, complete healing was somewhat slower than it was in animals with linear fracture. 2) Microangiography: The nutrient artery was normal down to the fracture site on the control side. On the experimentalside. the nutrient artery remained normal down to the most proximal point of the intramedullarynail. As healing progressed, circulation increased by development of new blood vessels at the cortex of the fracture site. This development was more pronounced on the experimental side. 3) Biochemistry: A week after experimental fracture the alkaline phosphatase levels in the blood of the experimental animals increased to more than twice that of the control animals. Serum phosphorus levels were one and one-half times higher than those of the control animals. No remarkable change was observed in serum calcium. There was no difference in bio'"" chemical studies between linear and segmental fractures. After the first week, all blood chemistries studied returned to normal. 4) Histology: Periosteal new bone formation was present at the end of one week. It was more advanced on the experimental side. At no time during the experiment did the intramedullary fixation side lag behind the control side in . healing. In a few cases the healing on the side with fixation was significantly ahead of the control side. Linear fractures healed more rapidly than segmental ones. The conclusion of the experimental study is that: 1) In no case did the use of intramedullary fixation delay bony union. 2) In a few cases the use of intramedullary fixation facilitated bony union.

Parapuat에 의한 肺胞鐵細症의 發生機轉에 關한 硏究

이상숙,정재홍,손태중 계명대학교 醫科大學 病理學敎室同門會 1987 樂山 鄭在泓 敎授 頌喜 紀念論文集 Vol.S No.-

This study was carried out to investigate the intricate mechanisms of intraalveolar fibrosis, leading to the alveolar structural remodeling, of rat lungs treated with paraquat. Sixty-three male Sprague-Dawley rats, maintained on a stock diet, weighing 200.0 gm, a verage, were divided into 4 experimental grohps. Group 1. Control group. Ten rats. Intraperitoneal injections of 2-4ml normal saline only. Group 2. Thirteen rats. Ten, 20, 25, 30 and 40 mg per kg of body weight was administered intraperitoneally. Animals were sacrificed 5 hours, 1 and 2 days after paraquat treatment. Group 3. Sixteen rats. Twenty, 25, 30 and 40mg per kg of body weight was administered to the animal, and animals died 2-5 days after para-quat administration. Group 4. Twenty-four rats. The same amount of paraquat was administered to the animal as in the group 2. Animals were sacrificed 1,2,6,8 and 10 weeks after paraquat treatment. Sacrificed animal lung was examined by gross, light-microscopic, immunohistochemical, ultrastructural observation, along with cellular and chemical analyses of bronchoalveolar lavage fluid. The results ,vere as follows: Grossy, 6 rats of chronic stage(1-10 weeks survival) developed multiple wedge-shaped scars on both lungs. These scars were situated mainly along the bronchial trees, blood vessels and subpleural ragions.Light microscopically, the salient features found of the chronic stage lungs were intraalveolar fibrosis. Intraluminal buds or polypoid masses projecting into the alveolar lumen and ducts. EIsewhere, loose connecti ve tissue masses were found to fuse together to alveolar wall, obliterating the alveolar spaces with resultant severe alveolar structural remodeling. Immunohistochemically, fibronectin was found in the center of intraalveolar buds and polypoid mass, projecting into the alveolar lumen, and in the adjacent proliferating alveolar macrophages. We regret to state that the attempt to measure the amount of fibronectin in the bronchoalveolar lavage fluid was a failure. Electron microscopically, the chronic stage lung revealed marked proliferation of both alveolar macrophages andfibroblasts in the alveolar spaces, the latter containing actin-like microfilaments and collagen fibers . arranged in bundles and spirals. In areas, myofibroblasts and smooth muscle cells also present. Cellular analysis of the bronchoalveolar lavage fluid in chronic stage lungs revealed no significant findings. I t can be concluded, therefore: That intraalveolar fibrosis of the paraquat treated lungs of the rat is probably mediated by intraalveolar migrations of the interstitial cells, the main task force being the connective tissue cells, passing through the defects created in the epithelial lining surface to its basement membrane, which were inflicted upon the alveolar wall by the paraquat toxicity. Fibronectin, released by activated alveolar macrophages, may be responsible for the migrations of fibroblasts and myofibroblasts into the alveolar spaces to form the intraalveolar fibrosis with subsequent alveolar structural remodeling.

사염화탄소투여로 인한 간세포상해와 간내 미세혈관계의 변화에 대한 주사전자현미경적 관찰

채종민 계명대학교 醫科大學 病理學敎室同門會 1987 樂山 鄭在泓 敎授 頌喜 紀念論文集 Vol.S No.-

lntracellular structures of hepatocytes and the hepatic microcirculation were studied in rats intoxicated with carbon tetrachloride by scanning electron microscopy. Liver tissues were treated by the DMSO cracking method and aldehyde-prefix osmium-DMSO-osmium method. A rapid, easy and reproducible method to prepare plastic corrosion casts of the hepatic . microcirculation employed Mercox CL-2B. The hepatocytes around the portal tracts showed minimal abnormalities. However, swollen foamy hepatocytes in intermediate zone and central zone had numerous vesicles and vacuoles, which correspond to dilated cisternae of rough endoplasmic reticula (RER) and fat droplets observed by transmission electron microscopy and aldehyde-prefix O-D-O method. And also, disorder of intrahepatic microcirculation was observed to be irregular-shaped sinusoids and distended space of Disse in acute hepatic injury. This abnormal microcirculation was well demonstrated three-dimensionally by scanning electron microscopy of corrosion casts.

사람 腦를 使用한 Thromboplastin 製造에 關한 實驗的 硏究

鄭在泓,宋文源 계명대학교 醫科大學 病理學敎室同門會 1987 樂山 鄭在泓 敎授 頌喜 紀念論文集 Vol.S No.-

1. The author has tried to prepare a standard thromboplastin for the determination of prothrombin time utilizing relatively fresh child human brain obtained at the time of autopsy, through acetone treatment. 2. A 13. 5 second thromboplastin extract, slightly slower than. the 12 second commercially available Difco product, was successfully prepared. 3. No influence on prothrombin time is’ noted when the thromboplastin solution is extracted from the powdered thromboplastin at the temperature, between 45 0C and 550 C. However, definite prolongation is observed when the extraction is made at above 600 C. 4. It appears that the optimal concentrations of powdered thromboplastin for the preparation of thromboplastin solution lie between 300 mg and 500 mg. 5. An attempt was made to prepare a slower acting thromboplastin sensitive at the level of therapeutic range, using rabbit brain and lung extract in equal parts, with uncertain success. 6. It is felt that the prospect of preparing a satisfactory fast acting standard thromboplastin is good, judging from the knowledge acquired through this experimentation. At this time, .however, we are unable to draw any specific conc1usion concerning the preparation of a sensitive slower acting thromboplastin from the rabbit.