Histological quantification of the tissue damage caused by PlasmaJet™ coagulator

The purpose of this study was to evaluate tissue damage caused by the PlasmaJet^TM coagulator in the uterus, ovary, and fallopian tube at different power settings in vitro and then to examine the damage caused in vivo. In vitro evaluation included prospective recruitment of six subjects undergoing hysterectomy with or without salpingo-oophorectomy. Tissue damage was evaluated histologically for power levels at 10%, 15%, and 20%, and for duration of 2 and 5 s at a clinically acceptable distance of 0.5 to 1 cm between the tip of probe and tissue. In vivo evaluation included 15 subjects undergoing hysterectomy with or without salpingo-oophorectomy. The most suitable power setting and duration of diathermy was decided from in vitro examination and applied on in vivo setting. Tissue damage was evaluated histologically. There was no significant difference seen in the depth and width of tissue damage in the in vitro specimens at different low power levels and duration of diathermy (P > 0.05). A setting of 20% power and duration of 5 s of diathermy was used therefore for in vivo setting. Mean ± SD depth (millimetres) of tissue damage in uterus, ovary, and fallopian tube were 0.63 ± 0.19, 0.61 ± 0.14, and 0.63 ± 0.18, respectively. Mean ± SD width (millimetres) of tissue damage in uterus, ovary, and fallopian tube were 4.66 ± 0.05, 4.05 ± 0.61, and 4.51 ± 0.77, respectively. Irrespective of tissue type, the average depth and width of tissue damage with application of Plasmajet^TM coagulator for 5 s at low power is 0.62 and 4.24 mm, respectively. It therefore appears to be a safe method of coagulation in vitro and in vivo at 20% power on gynaecological tissues.     

Growth of human glioblastomas as xenografts in the brains of athymic rats

BACKGROUND: We have developed xenografts of human glioblastoma (GBM) and established the baseline growth parameters and histopathological features of these tumors. MATERIALS-METHODS: Cells from 4 different human GBM cell lines were injected into the right caudate-putamen of brain in athymic rats. We measured tumor weights and the estimated survival time of each rat. RESULTS-CONCLUSION: U-251 MG and U-87 MG cells produced solid intracerebral tumors with a 100% tumor take rate, while SF-767 and SF-126 cells did not grow in the brains of athymic rats. Under the conditions employed, U-87 MG tumors grew faster than U-251 MG tumors, but both types of tumors exhibited reproducible growth characteristics from animal to animal. There was heterogeneity in the growth characteristics and histologies between the 2 tumor types, indicating that these tumor models might be useful for simulating some of the heterogeneity that occurs between GBM in humans.     

Effect of liquid depth on the synthesis and oxidation of nitric oxide in macrophage cultures

The effect of liquid depth on the synthesis of NO and O(2)(-) was studied in murine macrophage-like RAW 264.7 cells activated by bacterial lipopolysaccharide and interferon-gamma. Rates of NO(2)(-) and NO(3)(-) accumulation were determined 8-11 h after stimulation. The rate of NO synthesis was computed by using a reaction-diffusion model to correct NO(2)(-) and NO(3)(-) accumulation for physical loss of NO, whereas O(2)(-) synthesis was equated with NO(3)(-) formation. Rates of O(2)(-) synthesis determined by a spectrophotometric (cytochrome c) assay were in good agreement with those from NO(3)(-) accumulation and showed production of O(2)(-) to be detectable immediately, in contrast to the approximately 6 h time lag for NO. The assumption that NO(2)(-) and NO(3)(-) are stable end products of the extracellular oxidation of NO by O(2) and O(2)(-), respectively, was supported by the fact that NO(2)(-) and NO(3)(-) concentrations remained constant in the presence of unstimulated cells or stimulated cells where NO synthesis was inhibited. Data were obtained for media depths ranging from 1 to 4 mm. The physical loss of NO was found to be quite significant, exceeding NO(2)(-) and NO(3)(-) accumulation by an order of magnitude at the smallest depth. The principal finding was that the rates of NO(2)(-) and NO(3)(-) accumulation each remained nearly constant over the 4-fold range of liquid depths. Because greater depths should greatly facilitate the trapping of NO as NO(2)(-), this implies that NO synthesis decreased markedly with increasing depth. In contrast, O(2)(-) synthesis remained approximately constant. Oxygen availability is likely to have affected NO synthesis, in that diffusional limitations will yield the lowest cellular O(2) concentrations when the liquid depth is greatest and NO synthesis is known to decrease when O(2) levels are reduced. Concentrations of NO near the cells were calculated to remain at approximately 1 microM for all conditions examined, suggesting that regulation of NO synthase activity by NO might also have mediated the effect of liquid depth.     

Cell-biologic and functional analyses of five new Aquaporin-2 missense mutations that cause recessive nephrogenic diabetes insipidus

Mutations in the Aquaporin-2 gene, which encodes a renal water channel, have been shown to cause autosomal nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin. Most AQP2 missense mutants in recessive NDI are retained in the endoplasmic reticulum (ER), but AQP2-T125M and AQP2-G175R were reported to be nonfunctional channels unimpaired in their routing to the plasma membrane. In five families, seven novel AQP2 gene mutations were identified and their cell-biologic basis for causing recessive NDI was analyzed. The patients in four families were homozygous for mutations, encoding AQP2-L28P, AQP2-A47V, AQP2-V71M, or AQP2-P185A. Expression in oocytes revealed that all these mutants, and also AQP2-T125M and AQP2-G175R, conferred a reduced water permeability compared with wt-AQP2, which was due to ER retardation. The patient in the fifth family had a G>A nucleotide substitution in the splice donor site of one allele that results in an out-of-frame protein. The other allele has a nucleotide deletion (c652delC) and a missense mutation (V194I). The routing and function of AQP2-V194I in oocytes was not different from wt-AQP2; it was therefore concluded that c652delC, which leads to an out-of-frame protein, is the NDI-causing mutation of the second allele. This study indicates that misfolding and ER retention is the main, and possibly only, cell-biologic basis for recessive NDI caused by missense AQP2 proteins. In addition, the reduced single channel water permeability of AQP2-A47V (40%) and AQP2-T125M (25%) might become of therapeutic value when chemical chaperones can be found that restore their routing to the plasma membrane.     

Endosonographic characteristics of the anal sphincter complex in primigravid Sri Lankan women

OBJECTIVE: To determine the endosonographic anatomy of the anal sphincter complex in primigravid Sri Lankan women. METHOD: This is an observational study of 95 primigravid women admitted to an antenatal ward. They were examined using anal endosonography and data from subjects without sphincter injury were analyzed to describe anal sphincter morphology. Individual anal canal components were measured at defined levels, and the relationship of individual measurements with age and body mass index was calculated. RESULTS: Ninety-three of 95 women had no anal sphincter damage. The puborectalis sling (mean 5.5 mm, S.D. 0.77) forms the upper most border of the sphincter complex. The mean width of the deep external sphincter was 4.3 mm (S.D. 0.61); that of the superficial external sphincter was 4.5 mm (S.D. 0.56); and that of the internal anal sphincter at the mid anal canal was 2.1 mm (S.D. 0.21). The intersphincteric space (mean 2.1 mm, S.D. 0.26) could be distinguished sonographically in all subjects. The mean width of the anterior ring of the subcutaneous component was 3.8 mm (S.D. 1.01). The perineal body was sonographically identified in 60%. CONCLUSION: A set of normal values for the anal sphincter components in Sri Lankan primigravid women was established.     

Human glioblastoma cell lines: levels of low-density lipoprotein receptor and low-density lipoprotein receptor-related protein

The status of the low-density lipoprotein (LDL) receptor and LDL receptor-related protein (LRP) in seven human glioma cell lines was evaluated to extend our knowledge of human glioblastoma multiforme tumor metabolism for future drug design. Cell lines SF-767, SF-763, A-172, U-87 MG, U-251 MG, U-343 MG, and SF-539 were used. Binding of 125I-labeled LDL to these cells at 4 degrees C was carried out to determine the number of LDL receptors on cells and the affinity of LDL for these receptors. The content of LRP was measured by immunoblotting. The presence of specific saturable LDL receptors was proven in six of the cell lines investigated. SF-767 cells revealed high-affinity LDL binding (equilibrium dissociation constant, Kd = 7 nM) and maximum binding capacity approximating 300,000 receptors/cell. Most of the remaining cell lines had relatively lower affinity (Kd = 38-62 nM) but also had very high numbers of receptors (128,000-950,000/cell). All cell lines exhibited LRP, but the expression was variable. The cell lines SF-539, U-87 MG, and U-343 MG were particularly rich in this protein. The data suggest that glioblastoma cells have high numbers of LDL receptors; however, there is considerable variation in binding affinity. Overall, this finding suggests that LDL receptors on glioblastoma cells could potentially be useful for targeting antitumor agents. LRP, a multifunctional receptor expressed on glioblastoma cells, also has the possibility for serving as a therapeutic target.     

The neuroretinal rim in descending optic atrophy

We studied seven human eyes in which the changes of the neuroretinal rim areas following complete loss of all axons could be measured planimetrically. The mean diminution of the neuroretinal rim area was 40% (range from 26–72%). The optic nerve pathology causing the loss of axons was in the posterior portion of the nerve and the optic disc was therefore not directly disturbed by the injuries. It appears that less than half of the neuroretinal rim is occupied by nerve fibers. Offprint requests to: S.M. Drance