伊文氏蓝染色对大鼠脑内肿瘤生物学标记方法的研究

目的研究在大鼠脑内肿瘤模型中生物学染色标记脑内肿瘤的方法。方法选择F344大鼠,移植体外培养的RG2胶质瘤细胞制作脑内肿瘤模型,移植18d后经大腿静脉注射伊文氏蓝(Evans blue,EB)染色肿瘤组织,探讨生物学染色对脑内肿瘤的标示作用和手术中对肿瘤和周围正常脑组织鉴别的意义。结果EB浓度在100mg/kg时,肿瘤组织染色成鲜明蓝色,同时大鼠的皮肤和所有器官都被染色成蓝色;当浓度低于10mg/kg时,仅染色肿瘤组织而皮肤和其他器官不被染色;肉眼可鉴别的最低静注浓度为1.25mg/kg。肿瘤组织的染色效果持续到静注后96h。血清中EB浓度在静注后3h内迅速减少,之后呈缓慢代谢过程,静注后24h减少到静注浓度的38%,静注后24h EB在血清和肿瘤组织中的浓度差达到最大值。染色后在手术显微镜下进行肿瘤切除手术操作较未染色对照组明显容易,手术后切片组织学观察肿瘤被完全切除。结论EB生物学染色标记脑内肿瘤效果可靠,手术中作为肿瘤的标示鉴别肿瘤和周围正常脑组织的界限有效。     

Developmental changes in the behavioral and autonomic effects of kappa opioid receptor stimulation of the midbrain periaqueductal gray

Kappa opioid receptors stimulation with U50,488 is known to modulate behaviors during the early postnatal period, but the specific neuroanatomical locus of many of these effects is unexplored. In the present study, we infused U50,488 into the midbrain periaqueductal gray (PAG) and investigated the effects of this drug on behavior and heart rate of 1-, 2-, and 3-week-old rats. U50,488 increased activity most potently in 1- and 2-week-old subjects. Ultrasonic vocalization (USV) production was increased in 1-week-old subjects, but not in 2- or 3-week-old pups. Heart rate changes were similarly seen in younger aged subjects. At 1 week, U50,488 decreased heart rate, but at 2 weeks it increased heart rate. There was no effect of this drug on heart rate at 3 weeks. At 1 week, USVs were more potently elicited from dorsal than lateral PAG infusion sites. No other site-specific effects within the PAG were seen. The age-related decline in behavioral effects elicited by U50,488 is consistent with other published reports, and to the extent that kappa receptor activity mediates infant separation responses, implicates the PAG as a modulator of those responses.     

Functional consequences of inhibition of nucleotide breakdown in rat vas deferens: a study with Evans blue

The effect of Evans blue on nucleotide breakdown, nucleotide-evoked contractions and electrically evoked contractions, overflow of ATP and overflow of tritium (after labelling with [³H]-noradrenaline) was studied in rat vas deferens. Pieces of vas deferens degraded 83 to 85% of added ATP, ADP and 2-methylthio ATP (all 100 M) over 30 min. Evans blue (100 M) reduced this degradation to 22 to 26%. Nucleotides elicited contraction with potency declining in the order , \-methylene ATP > 2-methylthio ATP > ATP > ADP. Evans blue (100 M) shifted the concentration-response curve of , \-methylene ATP to the right and increased the maximum. Concentration-response curves of ATP, ADP and 2-methylthio ATP, in contrast, were shifted to the left and responses were much potentiated. In the presence of Evans blue, the rank order of potency was ATP > 2-methylthio ATP > , \-methylene ATP > ADP. Electrical field stimulation (100 pulses at 10 Hz) elicited contraction and an overflow of tritium and ATP. Evans blue (100 M) did not alter the contraction and the evoked overflow of tritium but increased 24-fold the evoked overflow of ATP. The results indicate that Evans blue may serve as an — albeit impure — ecto-nucleotidase inhibitor in functional experiments. Such experiments demonstrate that the low potency of ATP (and also ADP and 2-methylthio ATP) in eliciting contraction, and the small size of the overflow of ATP upon sympathetic nerve stimulation, are due to rapid breakdown.     

Concomitant blockade of P2X-receptors and ecto-nucleotidases by P2-receptor antagonists: functional consequences in rat vas deferens

In order to assess the consequences of a concomitant blockade of P2X-receptors and ecto-nucleotidases, effects of 13 P2-receptor antagonists were investigated on contractions of the rat vas deferens elicited by α,β-methylene ATP (α,β-MeATP) and ATP and on the removal of ATP from the incubation medium by vas deferens tissue. Increasing concentrations of all antagonists reduced and finally abolished contractions elicited by α,β-MeATP (3 μM), with IC₅₀-values ranging from 1.1 to 100 μM. Pyridoxalphosphate-6-azophenyl-2’,4’-disulphonate (PPADS), 6-azophenyl-4-amino-5-hydroxy-naphthalene-1,3-disulphonate (NH02), 4,4’-diisothiocyanatostilbene-2,2’-disulphonate (DIDS) and uniblue A also progressively reduced and finally abolished contractions elicited by ATP (1 mM). 8,8’-[Carbonylbis(imino-3,1-phenylenecarbonyl-imino)]-bis-(1,3,5-naphthalenetrisulphonate) (NF023), sura- min, pyridoxalphosphate-6-azophenyl-2’,5’-disulphonate (iso-PPADS), trypan blue and reactive blue 19, in contrast, caused only partial blockade, by 34–43% maximally; reactive blue 2 and reactive red 2 had no effect; and 6,6’-(1,1’-biphenyl-4,4’-diylbisazo)-bis-4-amino-5-hydroxy-naphtha-lene-1,3-disulphonate (NH01) and Evans blue even enhan- ced the response to ATP. For antagonists causing full or partial inhibition, the IC₅₀-values against ATP were close to those against α,β-MeATP. All antagonists attenuated the removal of ATP, with IC_25%-values ranging from 0.8 μM to >320 μM. The results confirm the frequent combination, in one antagonist molecule, of P2-receptor blockade and blockade of ecto-nucleotidases. This dual action underlies the effect of such compounds on contractions of the vas deferens elicited by ATP which, for certain substances (e.g., suramin, reactive blue 2), can be explained by a simple model in which the antagonist simultaneously blocks the degradation of ATP and a single contraction-mediating receptor (P2X₁). Several observations, however, do not conform with this model, and the existence of multiple contraction-mediating receptors for ATP or multiple, pharmacologically distinct ecto-nucleotidases has to be considered. Received: 23 October 1998 / Accepted: 11 January 1999     

Technetium-99m labeling of a molecule bearing easily reducible groups

Chemical stability of the naphthol-azo dye Evans Blue (EB) was examined in the presence of acidic stannous chloride (SnCl₂), with a view to preparing an instant cold kit. EB was found to be reactive toward this reducing agent, yielding the metal-chelating molecule 1,7-diamino-8-naphthol-2,4-disulfonic acid at high acidity and high stannous concentrations. This reduction reaction was undesirable in the cold kit preparation. The conditions were determined where reduction was inhibited, at pH = 5.0 and with a mole ratio of EB to SnCl₂ = [10:1], effecting the facile preparation of stable cold kits. Successful ^99mTc-labeling of an EB cold kit using these conditions resulted in the desired product with 98% radiochemical purity. Based on the radiolabeling efficiencies of chosen model compounds, it was rationalized that ^99mTc metal predominantly coordinated with the 1-amino-8-hydroxy groups in the EB molecule to form ^99mTc-EB.     

Catastrophic relapse of Evans syndrome five years after allogeneic BMT notwithstanding full donor chimerism. Terminal hemolytic-uremic syndrome

A patient with severe Evans syndrome received an allo-BMT from his HLA-identical sister on November, 2000. Full marrow and blood donor chimerism were achieved only after 5 donor lymphocyte infusions (DLI), and coincided with complete clinical remission and disappearence of auto-antibodies. Five years later, hemolytic anemia recurred with rapid increase of serum bilirubin to over 50 mg%: he responded to combined therapy, but died on day +17 from admission of an acute hemolytic uremic syndrome (HUS). All circulating blood cells, including erythrocytes, were 100% donor. Ex vivo cultured and expanded T and B cells from the peripheral blood were also 100% donor. The supernatants from B cell cultures, containing either IgM or IgG, did not react with a panel of erythrocytes. Thus in this typical autoimmune disease with a predominant B cell pathogenesis the donor immune system resulted “innocent of autoimmunity”. The persistence of long-lived recipient autoreactive plasma-cell lines in survival niches, still producing autoantibodies, may be hypothesized for this and similar cases. The postulated graft-versus-autoimmunity (GVA) effect was apparently not sufficient to eradicate autoimmunity in this patient.     

Effects of Lipopolysaccharide on the Blood-Brain Barrier Permeability in Prolonged Nitric Oxide Blockade-Induced Hypertensive Rats

The authors investigated the effects of lipopolysaccharide (LPS) on the blood-brain barrier (BBB) integrity and the activity of astrocytes during the N^w-nitro-L-arginine methyl ester (L-NAME) hypertension followed by angiotensin (ANG) II in rats. They measured the changes in the BBB permeability using the Evans blue (EB) dye and concomitantly in the levels of TNF-a, IL-1b, and IL-6 in serum and nitric oxide in plasma. The authors performed two tight junction-specific proteins, zonula occludens-1 and occludin, and glial fibrillary acidic protein, by using immunohisto-chemical method. The serum levels of TNF-α, IL-1β, IL-6, and the plasma level of nitric oxide significantly increased in LPS-treated rats (p < .01). The EB dye extravasation increased in cerebellum (p < .001) and diencephalon (p < .05) of L-NAME plus ANG II-treated animals. However, LPS reduced the increased EB dye extravasation in the brain regions of L-NAME-induced hypertensive rats treated with ANG II (p < .001). In L-NAME, there was a considerable loss of staining in both zonula occludens-1 and occludin. Staining for zonula occludens-1 and occludin was highly intensive in animals treated with LPS. Glial fibrillary acidic protein staining was seen in a few astrocytes in brains of L-NAME-treated animals. However, this staining showed an increased intensity in the brain sections of animals treated with LPS. This study indicates that, in L-NAME hypertensive rats, ANG II leads to an increase in the extravasation of EB dye to brain as a result of decreased activity of tight junction proteins and astrocytes, and LPS could significantly attenuate the EB dye transport to the brain through the increased activity of tight junction proteins and astrocytes.     

Chronic sleep loss disrupts blood–testis and blood–epididymis barriers,and reduces male fertility

Sleep loss increases blood–brain barrier permeability. As the blood–brain barrier and the blood–tissue barriers in the reproductive tract (blood–testis and blood–epididymis barriers) share common characteristics, we hypothesized that sleep restriction may also modify their barrier function. Previous reports showed that sleep loss decreased sperm viability and progressive fast mobility, which may be a consequence of altered blood–testis and blood–epididymis barrier. Therefore, we quantified changes in blood–testis and blood–epididymis barrier after sleep loss and related them to male fertility. Adult male Wistar rats were sleep restricted using the multiple‐platform technique in a protocol of 20 hr daily sleep deprivation plus 4 hr of sleep recovery in the home‐cage. At the 10th day, barrier permeability assays were performed with Na‐fluorescein, 10 kDa Cascade blue‐dextrans and Evans blue, and the expression of tight junction proteins, actin and androgen receptor was quantified. At the 10th day of sleep restriction and after sleep recovery days 1–7, males were placed with sexually receptive females, sexual behaviour was tested, and the percentage of pregnancies was calculated. Sleep restriction increased the barrier permeability to low‐ and high‐molecular‐weight tracers, and decreased the expression of tight junction proteins, actin and androgen receptor. Concomitantly, sleep restriction reduced the percentage of ejaculating males and the number of pregnancies. Sleep recovery for 2–3 days progressively re‐established fertility, as indicated by a higher percentage of ejaculating males and impregnated females. In conclusion, chronic sleep loss alters fertility concomitantly with the disruption of the blood–tissue barriers at the reproductive tract, the mechanism involves androgen signalling.     

Unusual complications after bone marrow transplantation for dyskeratosis congenita

Dyskeratosis congenita (DC) is a rare inherited disorder often associated with aplastic anaemia. We report the cases of five boys transplanted with an HLA-identical related donor for severe aplastic anaemia (SAA) associated to DC; in all cases successful engraftment was observed. Three patients died 2–8 years after bone marrow transplantation (BMT) with signs of endothelial cell damage syndrome (kidney microangiopathy and liver veno-occlusive disease). Another boy died 1 year after BMT from Evans syndrome and invasive aspergillosis. One boy currently presents anaemia, polyarthritis of unknown origin, pulmonary fibrosis and gut malabsorption 7.5 years after BMT. SAA associated with DC can be successfully treated by allogeneic BMT. However, these early and late complications observed are very unusual after BMT and probably reflect the association of transplanted-related factors, evolution of the underlying disease, and increased sensitivity of endothelial cells. Modified conditioning approaches, advances in supportive care and surveillance of these unusual complications offer the possibility of improved outcome for these patients.