Ultrastructural changes in the blood-brain barrier of mice infected with Plasmodium berghei.

Mice infected with the rodent malaria parasite Plasmodium berghei exhibit ultrastructural changes of the blood-brain barrier during the course of infection. Firm adherence including cellular interdigitation of infected cells or leucocytes and even clusters of cells to the vascular-endothelial lining is repeatedly observed early during infection. Ghosts and membrane remnants can be found engulfed in the surface of the endothelial cells. Frequently leucocytes migrate between endothelial cells and even cause a lift off and degeneration of these cells. In addition, endothelial cells exhibit increased pinocytotic activity, many irregular cytoplasmic extensions and even phagocytic activity. These changes are associated with degenerative changes in the basement membrane. Swelling and deposition of collagen-like fibres and even loss of fragments of basement membrane is observed. In some places fingerlike extensions of pericytes passed through the basement membrane and contacted or even bulged into the cytoplasm of endothelial cells. Ballooning and even coalescence of perivascular astrocytes was observed and contributed to the appearance of a perivascular oedematous space. The observed changes indicate a progressive deterioration of the blood-brain barrier eventually leading to endothelial lesions and hemorrhage.

Oxygen tensions in two human tumor cell lines grown and irradiated as multicellular spheroids.

Cells from two human cell lines were irradiated both as multicellular tumor spheroids (MTS) and in monolayer culture. Radiation response of MTS was quantified in terms of specific growth delay and proportion cured, and as clonogenic cell survival for monolayer cells. Radiation was applied either as a single or as a split dose with time intervals of 1, 2, and 4 h to determine the rate of sublethal damage repair. Using as endpoint the fraction of MTS cured at an iso-effect level, in MTS of NB-100 neuroblastoma cells repair of sublethal damage was complete within 1 h, whereas in MTS of HN-1 squamous cell carcinoma cells there was still some unrepaired damage left. At a larger dose for NB-100 MTS the repair curve showed a similar shape as for HN-1 spheroids. Using as endpoint specific growth delay, no difference in repair between the various time intervals was observed. In monolayer cells from both cell lines sublethal damage was not fully repaired in the time intervals used. Polarographic microelectrode measurements of oxygen tension inside MTS showed a marked difference in steepness of oxygen tension profiles between MTS from both cell lines. In HN-1 squamous cell carcinoma MTS with diameters up to 500 microns the central pO2 amounted to about 100 Torr, whereas in NB-100 neuroblastoma MTS with the same diameters central pO2-values lower than 30 Torr were observed. NB-100 MTS were irradiated with doses of 5 and 10 Gy gamma rays and subsequently the oxygen tension was measured 1 and 5 h after irradiation. A reoxygenation effect could not be observed, either after single dose or after split dose irradiation. If spheroids may be regarded as a suitable model for tumor responses in vivo, the results from these experiments indicate that reoxygenation is a process eluding polarographic measurements, or that no dramatic changes in oxygen tension are to be expected shortly after high single doses or early in a fractionation scheme.

Morphological characteristics of intracerebral arterioles in clinical (Plasmodium falciparum) and experimental (Plasmodium berghei) cerebral malaria.

Spastic constriction of intracerebral arterioles was identified in clinical (P. falciparum) and experimental (P. berghei) cerebral malaria. Morphological criteria were used to characterize pathologically spastic constriction of arterioles. The significance of spastic constriction of intracerebral arterioles for microcirculatory disturbance in relation to development of cerebral malaria is discussed.

A cytochemical study of cerebrovascular lesions in mice infected with Plasmodium berghei.

Mice with a Plasmodium berghei infection exhibit morphological and cytochemical changes in the blood-brain barrier. Changes in activity and localization of alkaline phosphatase and adenosine triphosphatase, enzymes with important functions in the maintenance of the blood-brain barrier, were observed. Changes in activity and localization of those enzymes in and near the endothelial cells of the microvasculature, concomitant with an increase in pinocytotic activity, and formation of irregular cytoplasmic extensions in these cells, as well as loosening of the basal lamina are indicative of a functional deterioration of the blood-brain barrier in the course of infection.

Radiosensitivity of human melanoma spheroids influenced by growth rate.

The radiosensitivity of human melanoma cell line BRO was investigated using the multicellular tumor spheroid system. By adding different concentrations of bovine serum to the tissue culture medium, two different growth rates could be obtained. Spheroids (200-250 microns) were irradiated with graded single doses of X rays (2-8 Gy). The radiation response was quantified using specific growth delay, clonogenic cell survival, and spheroid cure. All three assays showed a growth rate dependent radiation response. At both growth rates the spheroid growth fraction and critical cell number were of comparable magnitude. There was a strong correlation between the radiation response of spheroid regenerating units and clonogenic cells from dispersed spheroids. Cell survival curves indicated a decreased ability to accumulate sublethal damage in fast growing multicellular tumor spheroids. From this study it appears that the intrinsic radiosensitivity of human melanoma cell line BRO cells in multicellular spheroids is modulated by intratumoral conditions.

Sublethal damage repair and radiosensitivity of human squamous cell carcinoma cells grown with different culture techniques.

In this study, cells of a human squamous cell carcinoma line, HN-1, were grown in monolayers and as multicellular tumour spheroids (MTS). Repair of radiation-induced damage was studied by irradiation with single and split doses of X rays (4-8 Gy). It was shown that the amount of sublethal damage that was repaired in this dose range was equal in cells growing in monolayers and as MTS. The radiosensitivity of spheroids, as expressed by spheroid "cure" dose, increased with increasing MTS diameter. It is postulated that, in MTS with no signs of hypoxia, radioresistance diminishes when MTS increase in diameter.

Radiosensitivity of different human tumor cells lines grown as multicellular spheroids determined from growth curves and survival data.

Five human tumor cell lines were grown as multicellular tumor spheroids (MTS) to determine whether multicellular tumor spheroids derived from different types of tumors would show tumor-type dependent differences in response to single-dose irradiation, and whether these differences paralleled clinical behavior. Multicellular tumor spheroids of two neuroblastoma, one lung adenocarcinoma, one melanoma, and a squamous cell carcinoma of the oral tongue, were studied in terms of growth delay, calculated cell survival, and spheroid control dose50 (SCD50). Growth delay and cell survival analysis for the tumor cell lines showed sensitivities that correlated well with clinical behavior of the tumor types of origin. Similar to other studies on melanoma multicellular tumor spheroids our spheroid control dose50 results for the melanoma cell line deviated from the general pattern of sensitivity. This might be due to the location of surviving cells, which prohibits proliferation of surviving cells and hence growth of melanoma multicellular tumor spheroids. This study demonstrates that radiosensitivity of human tumor cell lines can be evaluated in terms of growth delay, calculated cell survival, and spheroid control dose50 when grown as multicellular tumor spheroids. The sensitivity established from these evaluations parallels clinical behavior, thus offering a unique tool for the in vitro analysis of human tumor radiosensitivity.

Repair of sublethal damage in two human tumor cell lines grown as multicellular spheroids.

Multicellular tumor spheroids (MTS) provide a suitable in vitro model to study radiation sensitivity of tumor cells. Two cell lines of human origin, obtained from a neuroblastoma (NB-100) and a squamous cell carcinoma (HN-1), were exposed to graded doses (4-9 Gy) of radiation with 18 MV photons. Radiation was applied either as a single or as a split dose with an interval of 6 hr to determine the extent of sublethal damage repair. Treated spheroids regrew at approximately the same growth rate as control multicellular tumor spheroids, preceded by a static or regression phase. Radiation response was quantified in terms of regrowth delay, expressed as the time needed for treated spheroids to obtain an 8-fold increase of the initial volume at the time of irradiation. Data obtained from regrowth delay analysis were used to calculate the extent of sublethal damage repair, showing for the squamous cell carcinoma line a fractionally higher capacity to repair sublethal damage than the neuroblastoma line. Repair increased with larger dose fractions in both cell lines. Our results show that multicellular tumor spheroids from the two cell lines used in this study are best applicable at relatively high total radiation doses. This makes multicellular tumor spheroids a suitable model for the in vitro evaluation of clinical treatment rationales such as hyperfractionation.

[Long-term experiences with lyophilized soft dura (Lyodura S) as a connective tissue substitute in pediatric surgery]. / Langzeiterfahrungen mit lyophilisierter Weichdura (Lyodura S) als Bindegewebsersatz in der Kinderchirurgie.

Between 1979 and 1985 plastic closures with lyophilised softened dura mater were performed in 45 children with the following indications: gastrochisis: 8; omphaloceles: 2; meningomyeloceles: 29; meningoceles: 3; diaphragmatic aplasia: 1; oesophageal atresia with tracheo-oesophageal fistula: 2. Infection as well as rejection of the implants were never observed. Mortality came up to 11%. Complications and causes of death are described. A follow-up was carried out postoperatively at semi-annual intervals. The scars showed good functional and cosmetic results.

Multicellular aggregates from human tumor cell lines for radiation studies.

Aggregates of human tumor cells are widely used in experimental studies on tumor responses to treatment. Only a limited number of human tumor cell lines are capable of forming spheroids. In this study cellular characteristics of 7 lung cancer and 4 bladder cancer cell lines are described with respect to their spheroid forming capacity. Comparisons were made with four reference lines known for their propensity to form growing aggregates. In the absence of vimentin expression no spherical aggregates were formed. Spherical aggregates were formed by one bladder and one lung cancer cell line, of which only the latter exhibited growth. Cellular factors influencing the ability of spheroids to increase in volume after spherical aggregation are not yet defined. Viability and clonogenicity of cells in aggregates are not the determinant of growth capacity. The growth rate of cell lines that exhibited growth is determined by tissue culture conditions and additives. Type of medium, percentage of foetal bovine serum and glucose concentration influenced the growth rate of spheroids. Since the response to radiation may be influenced by the growth rate of the tumors, manipulation of tissue culture medium composition offers the possibility of testing the influence of growth rate on the radiation response of one type of spheroids.

[Blood vessel replacement by new large (over 6 mm) and small (to 1.5 mm) lumen vascular prostheses]. / Der Blutleiterersatz durch neue gross- (über 6 mm) und kleinlumige (bis 1.5 mm) Gefässprothesen.

Histologic studies on 1,672 functionally implanted synthetic vascular grafts of various fabrics revealed 1. an industrially coated large-porous textile prosthesis to heal faster and without less complications than conventionally preclotted ones, 2. a neointimal endothelium to regenerate only from the vascular stumps, 3. the fate of neointima and of in vitro seeded endothelium to depend on the type of the vascular graft and 4. a new type of fine fibrillar vascular (polyurethane) microprostheses to have functioned in clinically relevant lengths with excellent results.

Immunological aspects of cerebral lesions in murine malaria.

The majority of male C57Bl/Rij mice died infected with Plasmodium berghei early in the second week. Death was closely correlated to collapse of the thermoregulation of the body, with perivascular oedema and petechial haemorrhages in the brain. Mice that did not show a collapse of thermoregulation (temperature drop below 30 degrees C) and survived for more than 2 weeks after infection did not show haemorrhages. Development of this syndrome (temperature below 30 degrees C; early death; haemorrhages) during infection depended on the presence of the spleen and was prevented by irradiation of the spleen or a timely treatment with dexamethasone, anti-T-cell serum or immune serum.

Histochemistry of cerebral lesions in mice infected with Plasmodium berghei.

The brains of mice with established symptoms of Plasmodium berghei erebral malaria were investigated histochemically and histologically. The activity of mitochondrial, lysosomal, glyco/glycogenolytic, hydrolytic and oxidizing enzymes as well as enzymes of the Krebs cycle and the pentose cycle was studied during the course of the infection. For comparison cryostate sections were also stained with haematoxylin and eosin, and according to Kluver-Barrera. Changes in enzyme activity, particularly of the vascular endothelium suggest a functional alteration of the blood-brain barrier which precedes the histochemically detectable lesions of the brain parenchyma. Decrease and total loss of enzyme activity in circumscript areas, also of ependymal cells were indicative of an early ischemic lesion. A population of small, non-phagocytozing, granuloma-like cells frequently accumulating in the frontobasal regions and in the subependymal zones were probably immature astrocytes. During early infection, these cells apparently fail to differentiate and turn to necrosis at the end of the second week. The results of this study support the concept of a triggering role of an initial vascular lesion and a functional breakdown of the blood-brain barrier in susceptible areas of the brain, in the pathogenesis of experimental malaria.

Volumetrical and morphological responses of human laryngeal squamous cell carcinoma xenografts treated with fractionated irradiation.

Xenografts of both primary human laryngeal squamous cell carcinoma and its metastases were irradiated with five daily fractions of 5.0 Gy. Tumor volume changes, morphology, mitotic index and mitotic figures were studied. Primary xenografts disappeared within 17 +/- 3 days. Grafts of metastases showed complete regression within 26 +/- 5 days, or regrowth after a delay period. Mitotic activity was higher in the grafts of metastases. The number of mitotic figures decreased and ceased within 14 days in the primary tumor, while some were still occasionally noted in the grafts of metastases. Telophase stages were practically absent after the first fraction. This study suggests that the response of metastases to radiation therapy is lower than the response of the primary tumor.

Alcohol-induced liver injury after jejunoileal bypass operation in rats.

The objective of this study was to investigate whether alcohol administration exerts a synergistic effect on jejunoileal bypass-induced liver dysfunction in rats. Male Wistar rats were subjected to 90% jejunoileal bypass or sham operation. For 10 weeks, subgroups were pair-fed either an alcohol-containing (36% of total calories) liquid diet or a liquid diet where alcohol was replaced isocalorically by starch. Alcohol feeding in rats with jejunoileal bypass increased hepatic triglyceride content about 6-fold as compared with bypassed rats receiving control diet. Neither jejunoileal bypass nor alcohol feeding led to significant changes in hepatic DNA and protein contents. Alcohol feeding increased cytochrome P-450 levels both in operated and in sham-operated rats. The administration of alcohol-containing diet decreased the activity of succinic dehydrogenase, the decrease being distinctly more pronounced in rats with jejunoileal bypass than in the sham-operated controls. Light microscopy revealed no significant morphological alterations in liver sections of rats fed the control diet after jejunoileal bypass or of rats receiving either the alcohol-containing diet or the control diet after sham operation. Alcohol feeding in bypassed rats, however, produced marked diffuse accumulation of fat, and regularly led to other histological abnormalities in the liver. These abnormalities included ballooning of hepatocytes and disarray of the trabecular structure of the liver lobule, hyalin inclusions resembling megamitochondria, single-cell necrosis and focal clustering of necrosis, increased number of mitotic figures, and infiltrates with inflammatory cells. The histological lesions of the liver of bypassed rats receiving alcohol exhibited no obvious zonal distribution. The results demonstrate that alcohol feeding to rats subjected to jejunoileal bypass leads to marked liver injury which mimics, at least in part, that of alcohol-induced liver disease in man. Rats subjected to jejunoileal bypass may, therefore, provide a new model for the study of alcoholic liver disease.

Development and applications of an animal model for human tumor xenografts.

Wistar rats treated with cyclophosphamide (4x 10 mg/kg), total lymphoid irradiation (9.0 Gy; dose rate 0.60 Gy/min) and cyclosporin A (15 mg/kg, daily, orally) developed a state of immune suppression permitting the growth of human tumor xenografts. Immunosuppression was monitored by lymphocyte counts, serum IgG determination. PHA and Con A lymphocyte-responses, proportion of B cells and histopathological studies of the lymphoid organs. The lymphocyte counts, IgG levels, PHA and Con A stimulation values remained severely depressed, during the period of cyclosporin A administration. Repopulation of the paracortical areas of the lymph nodes and the peri-arteriolar sheaths of the spleen did not occur, neither the reconstruction of the germinal centers in these organs. The thymus underwent severe atrophy. Seven of eight different types of human tumors were successfully xenografted in the immunomodified rat. The xenografted tumors maintained their original morphologic features and the mitotic rate did not change during subsequent transplantations.

Small bowel transplantation in the rat: the adverse effect of increased pressure during the flushing procedure of the graft.

The influence of the pressure during the flushing procedure on the results of the subsequent transplantation was investigated in a model of heterotopic functional small bowel transplantation in the outbred Wistar rat. In this model insufficient small bowel preservation will result in a lethal shock, soon after revascularization. If the size of the small bowel graft was reduced to a 15-cm segment of the proximal jejunum, improper preservation did not lead to lethal shock, but to histological changes of the graft. The influence of high flushing pressure (80 cm), medium flushing pressure (50 cm), low flushing pressure (35 cm), and no flushing procedure at all, was investigated in different groups of rats. Flushing was performed by gravity using chilled Ringer's lactate solution (4 degrees C), buffered with NaHCO3 at a pH of 7.4. The results were evaluated histologically as well as clinically. Results indicated flushing of the graft to be superior to a nonflushing procedure. A flushing pressure of 80 cm and even 50 cm proved to be harmful to the jejunal graft. Flushing pressure of 35 cm resulted in successful segmental (jejunal) and subtotal (minus 5- to 7-cm distal ileum) small bowel transplantation. However, survival of a total small bowel graft could not be achieved with the optimal flushing procedure indicating that the functional integrity of a small bowel graft also depends on conditions other than the method of short-term preservation.

Role of macrophages in the pathogenesis of endomyocardial fibrosis in murine malaria.

C57B1/Rij mice developed progressive endocardial oedema and extensive endocardial thrombosis with a predilection for the right half of the heart in the course of a lethal Plasmodium berghei infection. Chemotherapy of an ongoing infection resulted in fibrosis of affected areas. Despite a close correlation between development of lesions and parasitaemia, parasitized erythrocytes were not usually present in the affected areas. Macrophages might play, however, an important role in the pathogenesis of the lesions. Early changes included sticking of macrophages to the endocardial endothelium, migration to subendothelial areas associated with leakage, and oedema. Subsequently, subendothelial infiltrates of lymphocytes, neutrophilic granulocytes, macrophages and fibroblasts were found. Moreover, endothelial lesions, sometimes associated with cell migration, were found to be plugged by microthrombi. Mural thrombi grew out eventually until death of the host or until chemotherapy cleared the infection.

[Need for randomizing microvascular surgical experiments for testing new prosthesis material, with special reference to personal surgical errors from a morphological viewpoint]. / Uber die Notwendigkeit der Randomisierung mikrovaskulärer chirurgischer Experimente bei der Uberprüfung eines neuen Prothesenmaterials unter Berücksichtigung eigener chirurgischer Fehler aus morphologischer Sicht.

In the examination of material involving two microvascular prostheses of different nature the need is discussed of randomising experimental series to achieve a self-critical control of operative dexterity in microsurgical techniques and to exclude eventual errors arising and influencing the results. A comparison was made of two polyurethane prostheses, one porous (n = 15), and the other covered internally with a smooth silicon layer (n = 15). They were implanted using microsurgical techniques into the infrarenal portion of the abdominal aorta in rats. The main criterion for successful implantation was a patency over a prolonged period of observation lasting a maximum of 407 days. The porous prosthesis showed the best results. Most frequent complications were early thrombosis and technical faults demonstrated by histology. Therefore, the distribution through prospective randomisation of good and bad results based on technical errors enhances the significance of material analysis.