Europium fluorescence to visualize N-butyl 2-cyanoacrylate in embolized vessels of an arteriovenous malformation swine model.

BACKGROUND AND PURPOSE: Standard tissue staining using the lipid dye Oil-Red-O has been previously applied to stain vessel specimens, which were embolized with a mixture of n-butyl 2-cyanoacrylate (NBCA) and oil (Lipiodol). That technique, however, results in nonspecific and nonquantitative staining that does not provide the necessary differentiation between NBCA and Lipiodol. We present an innovative staining procedure that quantifies NBCA within treated tissues. METHODS: An arteriovenous malformation (AVM) model in swine was used to evaluate the polymerization characteristics of various ratios of Lipiodol/NBCA/glacial acetic acid (GAA) mixtures. To determine the depth of NBCA penetration within the AVM model and to characterize the polymerization patterns of various mixtures within the vessel, histologic cross- and longitudinal sections were prepared for microscopy. These paraffin-embedded tissue sections were stained with a europium aryl-beta-diketone complex (TEC) to improve differentiation between NBCA and Lipiodol. Quantification of NBCA and Lipiodol within the lumen of rete cross-sections was accomplished using image analysis software to determine percent luminal area occluded by embolization. RESULTS: Upon application of TEC, intense europium fluorescence was seen when the tissue samples were excited by low-power UV light (excitation at 365 nm; emission at 614 nm). The area of europium intensity within the lumen corresponded to NBCA concentration, and addition of GAA aided the NBCA distribution throughout the lumen without affecting fluorescence intensity. It was seen that NBCA could be easily differentiated from Lipiodol and that quantification could be readily performed on these sections because of the improved differentiation. For the case of a 50:50 (vol. %) mixture with an added 20 microL of GAA, luminal area distribution of Lipiodol, NBCA, and blood products was 42.6 +/- 3.5%, 33.8 +/- 5.7%, and 23.7 +/-2.7%, respectively. CONCLUSION: The rare earth metal europium, when added as a fluorescent chelate compound to histologic tissue sections, allowed for differentiation between NBCA and Lipiodol with good detail. These results have facilitated further characterization of NBCA polymerization for the use of AVM embolization.

Inhibition of nitric oxide but not prostacyclin prevents poststenotic dilatation in rabbit femoral artery.

BACKGROUND: Poststenotic dilatation (PSD) occurs in a low-pressure region where recirculation eddies oscillate in size during the cardiac cycle. NO may be an important mediator of PSD. METHODS AND RESULTS: Femoral arteries of 7 adult male New Zealand White rabbits were stenosed bilaterally to achieve a diameter reduction of 70. 9+/-6.7% (n=14). At the time of stenosis, the adventitia of one of the arteries was coated with 1 mmol/L of NG-nitro-L-arginine methyl ester (L-NAME) in 22% (wt/vol) Pluronic gel, while the contralateral vessel was coated with gel without L-NAME. In stenosed femoral arteries that were treated with gel without L-NAME, a maximum PSD of 30.99+/-7.92% (n=7) was observed in polymer casts at 3 days relative to the mean proximal diameter of 1.57+/-0.25 mm at a position 12 mm upstream of each stenosis. In contrast, the vessels treated with L-NAME exhibited a maximum PSD of only 7.16+/-8.81% (n=7) relative to the mean proximal diameter of 1.55+/-0.16 mm. L-NAME caused a 76. 9% reduction (P<0.001, n=7) of PSD. Similarly, NG-monomethyl-L-arginine 1 mmol/L and NG-nitro-L-arginine 10 micromol/L attenuated PSD by 57.5% (P<0.001, n=6) and 63.9% (P<0.05, n=6), respectively. Indomethacin 10 micromol/L caused no reduction in PSD. Arterial rings obtained from the poststenotic region were more sensitive and responsive to acetylcholine than those obtained proximal to the stenosis. CONCLUSIONS: NO, but not prostacyclin, is a major mediator of PSD.

A new model of radiation-induced myelopathy: a comparison of the response of mature and immature pigs.

PURPOSE: The development of an experimental model of radiation-induced myelopathy in the pig which would facilitate the study of the effects of clinically relevant treatment volumes. METHODS AND MATERIALS: The effects of local spinal cord irradiation, to a standard 10 x 5 cm field, have been evaluated in mature (37-42.5 weeks) and immature (15.5-23 weeks) pigs. Irradiation was with single doses of 60Co gamma-rays at a dose-rate of 0.21-0.65 Gy/min. The incidence of paralysis was used as an endpoint. RESULTS: Irradiation of mature animals resulted in the development of frank paralysis with animals showing combined parenchymal and vascular pathologic changes in their white matter. These lesions, in common with those seen in patients, had a clear evidence of an inflammatory component. The latency for paralysis was short, 7.5-16.5 weeks, but within the wide range reported for patients. However, it was shorter than that reported in other large animal models. The ED50 value (+/- SE) for paralysis was 27.02 +/- 0.36 Gy, similar to that in rats taking into account dose-rate factors. The irradiation of immature pigs only resulted in transient neurological changes after doses comparable to those used in the mature animals, ED50 value (+/- SE) 26.09 +/- 0.37 Gy. The reasons for these transient neurological symptoms are uncertain. CONCLUSION: A reliable experimental model of radiation-induced myelopathy has been developed for mature pigs. This model is suitable for the study of clinically relevant volume effects.

Cytotoxic immigration of granulocytes into megakaryocytes as a late consequence of irradiation.

The immigration of neutrophilic granulocytes into megakaryocytes was studied in the bone marrow of normal and X-irradiated beagles under various exposure conditions. Two groups of dogs received homogeneous total-body irradiation. One group received a dose of 1.6 Gy and the other received a dose of 2.4 Gy (midline tissue). A third group was irradiated from the left side of the body only. This exposure resulted in an inhomogeneous total-body irradiation (entrance dose 3.8 Gy, exit dose 0.9 Gy). A fourth group of animals received partial-body irradiation with a dose of 11.7 Gy delivered to the anterior two-thirds of the body, thereby subjecting about 70% of the hemopoietic marrow to irradiation. Dogs of a fifth group remained unexposed to irradiation and served as controls. The marrow was analyzed in sections of the ribs approximately 1 year after irradiation. The total number of megakaryocytes in one section was evaluated. The number of megakaryocytes showing granulocytes in their cytoplasm was determined and expressed as a percentage. This phenomenon can be explained as cytotoxic immigration of granulocytes into megakaryocytes. It was observed in approximately 1-2% of the megakaryocytes in the marrow of normal dogs. One year after irradiation the value increased to 10-26%. It was observed that neutrophilic granulocytes penetrated only into the large mature megakaryocytes in which the nuclei were mostly pyknotic. This phenomenon may be considered as a late effect of irradiation.

Development of blood vessel-related radiation damage in the fimbria of the central nervous system.

The identification problem of the dose-limiting tissue component was investigated in the CNS of rats. Moderate single doses of radiation, ranging from 20 to 25 Gy were applied to the brain of adult female rats. The sequence of events was analyzed by scoring a series of morphological changes in one of the white matter structures that appears to represent a sensitive location, that is the fimbria hippocampi. The previously defined "Tissue Injury Unit", characterized by a dilation of the blood vessel lumen, a thickening of the blood vessel wall, an enlargement of endothelial cell nuclei, and a hypertrophy of the adjacent astrocytes which represents a combined score of four different, but related histological changes, proved to be slightly more sensitive and responsive than the earliest recognizable changes in the neurological structures, that is demyelination. In addition, the incidence of demyelination could be expressed as a function of the intensity of the "Tissue Injury Unit". These findings can be interpreted as an additional indication that blood vessel changes and the hypertrophy of the perivascular astrocytes precede degenerative changes in the white matter of the CNS after moderate doses of X rays.

[The stem cell system of hematopoiesis: physiological and pathophysiological concepts]. / Das Stammzellsystem der Hämatopoese: physiologische und pathophysiologische Grundlagen.

It is the purpose of this review to describe the physiological as well as the pathophysiological principles of the hematopoietic stem cell system. The concept of hemopoietic stem cells has a long history which is now understood on the basis of its embryogenesis and after collecting extensive experimental and clinical experience using stem-cell transplantations as a means to restore hematopoietic function of the bone marrow after appropriate conditioning. The hemopoietic stem cells cannot be distinguished by light microscopy from "lymphocytes" considered to be a heterogeneous group of mononuclear cells. These stem cells can respond to specific regulatory factors with specific differentiation and proliferation, and are very radiosensitive and resistant to cryopreservation. The system responds to perturbations in a manner characteristic for feed back regulation and is bound in its physiology to an intact stromal matrix.

Hemodynamics alter arterial low-density lipoprotein metabolism.

We have investigated the role of hemodynamic factors on low-density lipoprotein transport and metabolism in the intact arterial wall. Freshly excised canine carotid blood vessels were exposed to well-defined pulsatile flow in vitro for continuous periods up to 20 hours. We chose to impose the following hemodynamic conditions on our test carotid arteries: normotension, hypertension (at physiologic flow conditions), and hypertension coupled with elevated flow of canine serum perfusate. In several experiments the effect of endothelial denudation was examined in carotid arteries exposed to normotensive pulsatile flow. A trapped ligand method was used for quantitating low-density lipoprotein uptake and metabolism in the arterial wall. The distribution of both intact and degraded low-density lipoprotein fractions was determined from measurements of radiolabelled low-density lipoprotein activity within thin radial sections of perfused arteries. Our results suggest that both hypertensive hemodynamic simulations exacerbate the uptake of low-density lipoprotein within the arterial wall (by a factor of three to nine). The percentage of low-density lipoprotein that undergoes irreversible degradation falls from 41% under normotensive conditions to below 30% when hypertensive conditions are imposed, indicating that degradative processes are not proportionally elevated with the accelerated influx. A similar pattern is observed for deendothelialized vessels.

Time- and dose-related changes in the white matter of the rat brain after single doses of X rays.

Following the local irradiation of the rat brain with single doses of 17.5-25 Gy of X rays, necrosis of the white matter was seen after a latent interval of greater than 26 weeks. At 39 weeks and 52 weeks after irradiation the incidence of necrosis was dose-related. The doses associated with a 50% incidence of necrosis in the white matter (ED50) at these times were 23.45 +/- 0.49 and 20.98 +/- 0.91 Gy, respectively. At both these times the incidence of necrosis was higher in the fimbria than in the capsula interna and the corpus callosum. This reflects a variation in the latency time for the appearance of necrosis. Necrosis occurs earlier in the fimbria. In the corpus callosum and the capsula interna the latency times for the appearance of necrosis were also dose-dependent. In the latent period prior to the onset of necrosis of the fimbria, a number of dose-related changes were seen in the vasculature and the associated astroglial cells. These changes, which included blood vessel dilation, blood vessel wall thickening, endothelial cell nuclear enlargement and the hypertrophy of perivascular astrocytes, were highly correlated and when combined appeared to represent a "unit of tissue injury". The incidence and severity of this "unit of tissue injury" apparently increased with time after irradiation until necrosis ensued. These dose-related vascular/glial changes were preceded by a reduction in the endothelial cell and vascular density. No early changes were seen in the number of glial parenchymal cells.

Degeneration and atrophy of the thymus of lethally irradiated dogs, rescued by transfusion of cryopreserved autologous blood leukocytes.

Dogs exposed to a fatal radiation dose of 12 Gy were rescued by transfusion of autologous blood leukocytes. A severe acute and long-lasting damage to the thymus was observed. The acute damage, as observed on the tenth day, consisted of a marked reduction in the number of lymphocytes, degeneration of Hassall's bodies, and hemorrhage. Long-term effects, observed several months after irradiation, were partial to total atrophy of the thymus. Regeneration, when it occurred, was limited to a few small isolated areas in which lymphopoiesis was supported by epithelial reticular cells. In contrast, the lymph nodes of all dogs had abundant cortical lymphopoiesis. The abundant hemopoiesis present in the marrow from the tenth day after irradiation until the end of the observation period should have provided sufficient circulating precursor cells to seed the thymus and regenerate the organ to the same extent as that observed in the other blood-forming organs. The impairment of lymphopoietic regeneration in the thymus seems to be due, therefore, to damage caused by irradiation on the specific stroma of the organ, which is not able to support such activity.

Dose-dependent and time-dependent changes in the choroid plexus of the irradiated rat brain.

A histological assessment has been made of both time- and dose-related changes in the choroid plexus after the local irradiation of the rat brain with single doses of 17.5-25 Gy of X rays. These investigations involved the serial killing of animals 1-52 weeks after irradiation and the quantitative and semiquantitative evaluation of histological sections. Counts of the relative number of cells in the choroid plexus of the lateral ventricles showed an atrophy of the epithelial layer after 13 weeks. However, this was not as marked as the reduction in the number of endothelial cells in the wall of blood vessels. Moreover, the epithelium had recovered by 39 weeks after irradiation, while the dose-related depletion in endothelial cells tended to be progressive. A highly correlated group of changes in the vascular-connective tissue was used to produce a numerical "factor". This represented a combined score of radiation damage which was both time- and dose-related. These data suggest that, as an expression of late radiation damage to the choroid plexus, the effect on the endothelium was more important than that to the epithelial cells.

Development and histogenesis of the thymus in dog. A light and electron microscopical study.

The development of the thymus was studied with histological, transmission electron microscopic (TEM) and histochemical methods in 100 dog fetuses (beagle), between day 19 of gestation and day 21 after birth. Thymus development could be divided in three stages: 1/Formation of epithelial palisades; 2/Initiation of lymphopoiesis; 3/Differentiation of the medulla and Hassall's bodies (HB). The epithelial anlagen were seen at day 23 of gestation showing the characteristic palisade structure of the endodermally derived epithelium. Ten days later the beginning of lymphoiesis and the reticularization of the epithelial cells could be seen. The first HB could be found at day 38 when cortical-medullary differentiation is recognized. The histochemical observation demonstrated a rich content of PAS positive coarse granules in the cytoplasm of reticulo-epithelial (RE) cells. On the other hand, the HB showed a diffuse PAS positive reaction. The ultrastructural investigations demonstrated the presence of desmosomes connecting RE cells to one another. Desmosomes were not found between RE and lymphocytes. The growth of the developing thymus into the mesenchymal matrix resulted in the lobulation of the organ by connective tissue cells and fibers. The first mast cells were seen at day 35 of gestation, most abundantly in the interlobular connective tissue (ICT) although a few were present in the cortex and somewhat more in the medulla, near the HB. At the end of development small groups of neutrophil cell precursors appeared in the ICT and the cortex. Cysts were not present up to day 21 after birth.

What kind of morphologically recognizable haemopoietic cells do we inject when doing foetal liver infusion in man?

There is no agreement, how to define the age of the embryo/foetus. From the 15th (fertilization) week onwards the whole foetal liver contains some 10(9) free haemopoietic cells. Before, and up to the 30th-34th weeks, the liver is the main site of haemopoiesis. The differential of embryonic smears (up to wk 9) differs from that of foetal ones. The first invasion of circulating primitive erythroblasts seeding in the liver (early 5th wk) is accompanied by the appearance of a lot of sinusoidal macrophages. Definitive erythropoiesis expands during the 6th-7th wks. Granulocytic representation peaks at 15-16 wks. Megakaryocytes are small and have few nuclei/nuclear lobes. Five to 8, or even more per cent of single haemopoietic cells were lymphoid-like cells in the 5th-6th wk liver smear. These cells precede the development of any lymphoid structure in the foetus. Ordinary lymphocytes amount to less than 2% between 10 and 18 wks, and reached 3% at wk 24. Percentage of dyserythropoietic nuclei in smears has been used to decide whether the injected cells could be regarded as 'physiological' cells. Ten out of 11 apparently healthy foetuses, delivered by hysterectomy/hysterotomy for maternal interest, aged 10 1/2 to 20 1/2 weeks, had mean 4.5% liver dyserythropoiesis. Extremely high dyserythropoiesis was associated with multilineage, instead of overwhelmingly erythroid haemopoiesis.

Variation of treatment conditions alters the outcome of fetal liver transplantation in dogs.

Fetal liver transplants (FLT) were carried out in 25 beagles under various conditions. Graft recipients were prepared with fractionated total body x-irradiation (TBI) of 3 X 6 Gy or 2 X 6 Gy and rescued with cryopreserved fetal liver cells (FLC) from 51- to 52-day-old or 43- to 46-day-old, DLA-identical siblings or DLA-haploidentical, homozygous half-siblings. In all groups, FLC grafts contained comparable numbers of granulocyte-macrophage progenitor cells. Initial engraftment was achieved in all dogs. However, low TBI dose and DLA haplotype disparity between donor and recipient were associated with graft failure in 1/3 and 2/9 recipients, respectively, within 10-16 days of treatment. Lectin-responsive host type lymphocytes circulated for more than 5 weeks, whereas bone marrow metaphases were always of donor sex. Reduced TBI dose and young donor age were associated with delayed granulocyte recovery. Moreover, circulating platelets and total lymphocytes as well as T and B-cell numbers and rose more slowly in recipients of immature FLC grafts than in the other groups. Delayed cutaneous hypersensitivity reactions were normal one year after FLT, whereas the IgM component of the hemagglutinin response to sheep red blood cells was depressed. In mixed leukocyte culture, chimeric lymphocytes were tolerant to host antigens. Nonetheless, clinical and histological signs compatible with low-grade graft-versus-host disease were recorded in 10 or 25 animals. Thus FLT in dogs could be carried out, even across DLA barriers, without severe graft-versus-host disease. However, a low pretransplant TBI dose, incomplete DLA match and young age of the fetal donor were associated with graft rejection and protracted restoration of hemopoiesis and immune functions.

Effects of local irradiation and i.v. methotrexate on brain morphology in rabbits: early changes.

Brain damage following cranial radiation therapy can be crippling or even life-threatening and has been studied in both patients and animals. An additional toxic effect of chemotherapy has been found in children, who died following brain irradiation and systemic chemotherapy for the treatment of acute lymphoblastic leukemia. To study the interaction of radiation and drugs on brain tissue, we treated rabbits with brain irradiation and/or i.v. methotrexate. For a period of up to 3 months following radiation therapy, brain morphology was compared in seven treatment groups. Weekly doses of methotrexate administered i.v. produced no brain damage. Histological examination showed myelin swelling and beading 14 weeks after fractionated brain irradiation with 24 Gy. Combination of brain irradiation and methotrexate produced additional hypertrophy of microglia and pyknosis of adventitial cells. In none of these groups, even after doses of 48 Gy brain irradiation, was calcification or brain necrosis observed during the first 14 weeks following irradiation.

Acute and long-term alterations in the granulocyte/macrophage progenitor cell (GM-CFC) compartment of dogs after partial-body irradiation: irradiation of the upper body with a single myeloablative dose.

The acute and long-term effects of a single dose of partial-body irradiation on the granulocyte/macrophage progenitor cell compartment were studied in dogs. A myeloablative dose of 11.7 Gy (dose rate 6.5 cGy/min) was given to the upper body which contains approximately 70% of the total bone marrow mass. The lower part of the body (pelvis, lower extremities and tail) was shielded by a lead box. In the non-irradiated bone marrow, the concentration of the GM-CFC/10(5) mononuclear cells was slightly decreased within the first 7 days and showed some fluctuations around the normal value for several weeks thereafter. In the irradiated bone marrow, virtually no GM-CFC could be detected on day 1 after exposure. Beginning on day 7, a continuous increase took place up to day 21 when the GM-CFC concentration reached between 25% (sternum) and 43% (humerus) of the initial value. No further increase took place up to day 80. Between day 120 and 380 a secondary increase was observed which reached near-normal bone marrow GM-CFC concentrations. The blood GM-CFC concentration first showed a strong depression followed by a transient increase between day 10 and 30. This coincided with GM-CFC normalization in the protected bone marrow as well as with the initial phase of regeneration in the irradiated sites. A prolonged secondary long-lasting depression between day 33 and 120 amounted to 20 to 50% of normal values. This depression was closely related to the stagnation in the GM-CFC recovery in the irradiated bone marrow sites. The GM-CFC concentration in the blood was found to be supranormal at day 380 when the bone marrow GM-CFC had recovered. The colony stimulating activity in the serum showed an increase within the first 20 days after exposure, that is, within the same interval the bone marrow GM-CFC concentration experienced the strongest alterations, and was inversely related to the changes in the blood granulocyte values.