Novel multi-linear quantitative brain volume formula for manual radiological evaluation of brain atrophy.

OBJECTIVES: The brain atrophy commonly occurs in elderly and in some childhood conditions making the techniques for quantifying brain volume needful. Since the automated quantitative methods of brain volume assessment have limited availability in developing countries, it was the purpose of this study to design and test an alternative formula that is applicable to all healthcare levels. METHODS: The multi-linear diagonal brain fraction formula (DBF) was designed from dimensions of brain relative to skull and ventricles. To test a developed formula, a total of 347 subjects aged between 0 and 18 years who had brain CT scans performed recruited and subjected to a systematic measurement of their brains in a diagonal brain fashion. RESULTS: Out of 347 patients evaluated, 62 subjects (17.8 %) were found to be cases of brain atrophy. The three radiological measurements which included sulcal width (SW), ventricular width (VW) and Evans Index (EI) were concurrently performed. SW and VW showed good age correlation. Similar tests were extended to diagonal brain fraction (DBF) and skull vertical horizontal ratio (VHR) in which DBF showed significant age correlation. CONCLUSIONS: The DBF formula shows significant ability of differentiating changes of brain volume suggesting that it can be utilized as an alternative brain fraction quantification method bearing technical simplicity in assessing gross brain volume. ADVANCES IN KNOWLEDGE: The designed formula is unique in that it captures even the possible asymmetrical volume loss of brain through diagonal lines. The proposed scores being in term of ratios give four grades of brain atrophy.

Valvular and Mural Endocardiosis in Aging Zebrafish (Danio rerio).

Endocardiosis or myxomatous degeneration of the cardiac valves is a well-described age-related change in humans and dogs. Lesions consist of polypoid nodular proliferations of loose extracellular matrix and valvular interstitial cells, most commonly affecting the mitral valve. This entity has not been previously described in fish. Herein we report the appearance, location, and occurrence of valvular and mural endocardiosis in a retrospective survey of aging laboratory zebrafish. Endocardiosis was present in 59 of 777 fish (7.59%), most commonly affecting the sinoatrial (34 fish; 57.6%) and atrioventricular (33 fish; 55.9%) valves. Lesions were more common in fish raised in recirculating water systems and fed commercial diets (52/230 fish; 22.6%) versus flow-through systems with fish fed semi-purified diets (4/234; 1.71%). Lesions were overrepresented in fish heterozygous for a mutant smoothened allele (34/61 fish, 55.7% vs 17/168, 10.1% wild type). There was no association between endocardiosis and intestinal carcinoids. Valvular endocardiosis is a significant age- and husbandry-related background finding in zebrafish and should be considered in the design and interpretation of research studies.

Primary intestinal and vertebral chordomas in laboratory zebrafish (Danio rerio).

Chordomas are uncommon neoplasms arising from notochord remnants, most commonly occurring in the axial skeleton. Extraskeletal soft tissue chordomas are rare primary tumors, and primary alimentary tract chordomas have not been reported. Herein we report 24 cases of spontaneous primary intestinal chordomas in zebrafish, as well as 9 spontaneous vertebral chordomas. Both intestinal and vertebral tumors showed invasive behavior, although more commonly in the latter. In all cases of primary intestinal chordomas, there was no axial or peripheral skeletal or other nonvisceral involvement. Although uncommon, intestinal chordomas represent a unique background lesion in aged zebrafish.

Activation of Sonic hedgehog signaling in neural progenitor cells promotes glioma development in the zebrafish optic pathway.

Dysregulation of Sonic hedgehog (Shh) signaling has been implicated in glioma pathogenesis. Yet, the role of this pathway in gliomagenesis remains controversial because of the lack of relevant animal models. Using the cytokeratin 5 promoter, we ectopically expressed a constitutively active zebrafish Smoothened (Smoa1) in neural progenitor cells and analyzed tumorigenic capacity of activated Shh signaling in both transient and stable transgenic fish. Transient transgenic fish overexpressing Smoa1 developed retinal and brain tumors, suggesting smoa1 is oncogenic in the zebrafish central nervous system (CNS). We further established stable transgenic lines that simultaneously developed optic pathway glioma (OPG) and various retinal tumors. In one of these lines, up to 80% of F1 and F2 fish developed tumors within 1 year of age. Microarray analysis of tumor samples showed upregulated expression of genes involved in the cell cycle, cancer signaling and Shh downstream targets ptc1, gli1 and gli2a. Tumors also exhibited specific gene signatures characteristic of radial glia and progenitor cells as transcriptions of radial glia genes cyp19a1b, s100ß, blbp, gfap and the stem/progenitor genes nestin and sox2 were significantly upregulated. Overexpression of GFAP, S100ß, BLBP and Sox2 was confirmed by immunofluorescence. We also detected overexpression of Mdm2 throughout the optic pathway in fish with OPG, therefore implicating the Mdm2-Tp53 pathway in glioma pathogenesis. In conclusion, we demonstrate that activated Shh signaling initiates tumorigenesis in the zebrafish CNS and provide the first OPG model not associated with neurofibromatosis 1.

Crosstalk of Ras and Rho: activation of RhoA abates Kras-induced liver tumorigenesis in transgenic zebrafish models.

RAS and Rho small GTPases are key molecular switches that control cell dynamics, cell growth and tissue development through their distinct signaling pathways. Although much has been learnt about their individual functions in both cell and animal models, the physiological and pathophysiological consequences of their signaling crosstalk in multi-cellular context in vivo remain largely unknown, especially in liver development and liver tumorigenesis. Furthermore, the roles of RhoA in RAS-mediated transformation and their crosstalk in vitro remain highly controversial. When challenged with carcinogens, zebrafish developed liver cancer that resembles the human liver cancer both molecularly and histopathologically. Capitalizing on the growing importance and relevance of zebrafish (Danio rerio) as an alternate cancer model, we have generated liver-specific, Tet-on-inducible transgenic lines expressing oncogenic Kras(G12V), RhoA, constitutively active RhoA(G14V) or dominant-negative RhoA(T19N). Double-transgenic lines expressing Kras(G12V) with one of the three RhoA genes were also generated. Based on quantitative bioimaging and molecular markers for genetic and signaling aberrations, we showed that the induced expression of oncogenic Kras during early development led to liver enlargement and hepatocyte proliferation, associated with elevated Erk phosphorylation, activation of Akt2 and modulation of its two downstream targets, p21Cip and S6 kinase. Such an increase in liver size and Akt2 expression was augmented by dominant-negative RhoA(T19N), but was abrogated by the constitutive-active RhoA(G14V). Consequently, induced expression of the oncogenic Kras in adult transgenic fish led to the development of hepatocellular carcinomas. Survival studies further revealed that the co-expression of dominant-negative RhoA(T19N) with oncogenic Kras increased the mortality rate compared with the other single or double-transgenic lines. This study provides evidence of the previously unappreciated signaling crosstalk between Kras and RhoA in regulating liver overgrowth and liver tumorigenesis. Our results also implicate that activating Rho could be beneficial to suppress the Kras-induced liver malignancies.

Glial cell line-derived neurotrophic factor (GDNF) expression and NMJ plasticity in skeletal muscle following endurance exercise.

Glial cell line-derived neurotrophic factor (GDNF) supports and maintains the neuromuscular system during development and through adulthood by promoting neuroplasticity. The aim of this study was to determine if different modes of exercise can promote changes in GDNF expression and neuromuscular junction (NMJ) morphology in slow- and fast-twitch muscles. Rats were randomly assigned to a run training (run group), swim training (swim group), or sedentary control group. GDNF protein content was determined by enzyme-linked immunosorbant assay. GDNF protein content increased significantly in soleus (SOL) following both training protocols (P<0.05). Although not significant, an increase of 60% in the extensor digitorum longus (EDL) followed swim-training (NS; P<0.06). NMJ morphology was analyzed by measuring α-bungarotoxin labeled post-synaptic end plates. GDNF content and total end plate area were positively correlated. End plate area decreased in EDL of the run group and increased in SOL of the swim group. The results indicate that GDNF expression and NMJ morphological changes are activity dependent and that different changes may be observed by varying the exercise intensity in slow- and fast-twitch fibers.

Epizootic neoplasia of the lateral line system of lake trout (Salvelinus namaycush) in New York's Finger Lakes.

This article documents an epizootic of inflammation and neoplasia selectively affecting the lateral line system of lake trout (Salvelinus namaycush) in 4 Finger Lakes in New York from 1985 to 1994. We studied more than 100 cases of this disease. Tumors occurred in 8% (5/64) of mature and 21% (3/14) of immature lake trout in the most severely affected lake. Lesions consisted of 1 or more neoplasm(s) in association with lymphocytic inflammation, multifocal erosions, and ulcerations of the epidermis along the lateral line. Lesions progressed from inflammatory to neoplastic, with 2-year-old lake trout showing locally extensive, intense lymphocytic infiltrates; 2- to 3-year-old fish having multiple, variably sized white masses up to 3 mm in diameter; and fish over 5 years old exhibiting 1 or more white, cerebriform masses greater than 1 cm in diameter. Histologic diagnoses of the tumors were predominantly spindle cell sarcomas or benign or malignant peripheral nerve sheath neoplasms, with fewer epitheliomas and carcinomas. Prevalence estimates did not vary significantly between sexes or season. The cause of this epizootic remains unclear. Tumor transmission trials, virus isolation procedures, and ultrastructural study of lesions failed to reveal evidence of a viral etiology. The Finger Lakes in which the disease occurred did not receive substantially more chemical pollution than unaffected lakes in the same chain during the epizootic, making an environmental carcinogen an unlikely primary cause of the epizootic. A hereditary component, however, may have contributed to this syndrome since only fish of the Seneca Lake strain were affected.

Short-term exercise increases GDNF protein levels in the spinal cord of young and old rats.

Neurotrophic factors may play a role in exercise-induced neuroprotective effects, however it is not known if exercise mediates changes in glial cell line-derived neurotrophic factor (GDNF) protein levels in the spinal cord. The aim of the current study was to determine if 2 weeks of exercise alters GDNF protein content in the lumbar spinal cord of young and old rats. GDNF protein was quantified via an enzyme-linked immunosorbent assay and Western blot. Immunohistochemical analysis localized GDNF in choline acetyltransferase (ChAT)-positive motor neurons and cell body areas were measured. Involuntary running in the young animals appeared to elicit the greatest increase in GDNF protein content (sixfold increase), followed by swimming (threefold increase) and voluntary running (twofold increase); however there was no significant difference between the modalities of exercise. Low-intensity running of the old animals significantly increased GDNF protein content in the spinal cord. Both young and old exercised animals showed a doubling in ChAT-positive motor neuron cell body areas. These results suggest that GDNF protein content in the spinal cord is modulated by exercise.

Glial cell line-derived neurotrophic factor protein content in rat skeletal muscle is altered by increased physical activity in vivo and in vitro.

Current evidence suggests that exercise and glial cell line-derived neurotrophic factor (GDNF) independently cause significant morphological changes in the neuromuscular system. The aim of the current study was to determine if increased physical activity regulates GDNF protein content in rat skeletal muscle. Extensor Digitorum Longus (EDL) and Soleus (SOL) hind limb skeletal muscles were analyzed following 2 weeks of involuntary exercise and 4 h of field stimulation or stretch in muscle bath preparations. GDNF protein content was measured via enzyme-linked immunosorbent assay (ELISA). Two weeks of exercise increased GDNF protein content in SOL as compared to sedentary controls (4.4±0.3 pg GDNF/mg tissue and 3.1±0.6 pg GDNF/mg tissue, respectively) and decreased GDNF protein content in EDL as compared to controls (1.0±0.1 pg GDNF/mg tissue and 2.3±0.7 pg GDNF/mg tissue, respectively). GDNF protein content in the EDL decreased following both field stimulation (56%±18% decrease from controls) and stretch (66%±10% decrease from controls). SOL responded to field stimulation with a 38%±7% increase from controls in GDNF protein content, but showed no change following stretch. Pre-treatment with α-bungarotoxin abolished the effects of field stimulation in both muscles and blocked the effect of stretch in EDL. α-bungarotoxin pre-treatment and stretch increased GDNF protein content to 240%±10% of controls in the SOL. Exposure to carbamylcholine decreased GDNF protein content to 51%±28% of controls in the EDL but not SOL. These results suggest that GDNF protein content in skeletal muscle may be controlled by stretch, where it may increase GDNF protein content, and membrane depolarization/acetylcholine (ACh) which acts to decrease GDNF protein content.

Histological and neurotrophic changes triggered by varying models of bladder inflammation.

PURPOSE: We determined whether bladder inflammation causes elevated expression of nerve growth factor by bladder parenchymal cells, leading to alterations in neurons innervating the bladder. To answer this question biochemical, histological and neuronal size data were obtained in rats following various experimental models of bladder inflammation. MATERIALS AND METHODS: Chemical (2.5% formalin), immune (lipopolysaccharide 2 x 104 cfu/ml.) and mechanical (chromic catgut) inflammation was evaluated at various times and compared to control bladders. Hematoxylin and eosin, and Giemsa staining was done to characterize inflammation and quantify mast cells in the bladder. Nerve growth factor protein and messenger RNA were assayed in the bladder and major pelvic ganglion using 2-site enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction, respectively. Retrograde axonal tracing was done to size bladder neurons in the major pelvic and dorsal root ganglia. RESULTS: All forms of inflammation increased bladder weight and produced diffuse hyperplasia, intramural edema, acute and chronic inflammatory cells, infiltration and mastocytosis. Generally bladder inflammation resulted in a 50% increase in nerve growth factor and 52% to 58% enlargement of peripheral neurons. CONCLUSIONS: Inflammation results in altered nerve growth factor content of the bladder, and morphological changes in sensory and motor neurons innervating the bladder. Such neuroplasticity may be a possible explanation for the association of bladder inflammation with long-term symptoms and pain after inflammation subsides.

Plasma androgen correlation, EROD induction, reduced condition factor, and the occurrence of organochlorine pollutants in reproductively immature white sturgeon (Acipenser transmontanus) from the Columbia River, USA.

White sturgeon (Acipenser transmontanus) support an active fishery in the Columbia River, but there is poor reproductive success within the impounded sections. The poor reproductive success has been attributed to hydroelectric development; however, water pollution could be a significant factor. White sturgeon plasma, liver, and gonad samples were collected from four Columbia River locations and a California aquaculture facility. Total length and weight of the fish were measured, and plasma samples were analyzed for testosterone (T), 11-ketotestosterone (KT), 17 beta-estradiol (E2), and vitellogenin. Liver samples were analyzed for chlorinated pesticides and polychlorinated biphenyls, ethoxyresorufin-O-deethylase (EROD) activity and histopathology. Gonads were examined histologically to assess sexual maturity and characterize any lesions. Significant differences by location existed for p,p'-DDE, EROD activity, and condition factor. Plasma T was negatively correlated with p,p'-DDE in males and females, and plasma KT was negatively correlated in males. These data indicate that pollutants could be adversely affecting white sturgeon in the Columbia River basin.

Fumonisin B1 promotes aflatoxin B1 and N-methyl-N'-nitro-nitrosoguanidine-initiated liver tumors in rainbow trout.

Laboratory studies have described the carcinogenicity of fumonisin B1 (FB1) in rodents and epidemiological evidence suggests an association between FB1 (a mycotoxin produced by Fusarium moniliforme) and cancer in humans. This study was designed to reveal in rainbow trout, a species with very low spontaneous tumor incidence, if FB1 was (i) a complete carcinogen, in the absence of an initiator; (ii) a promoter of liver tumors in fish initiated as fry with aflatoxin B1 (AFB1); and (iii) a promoter of liver, kidney, stomach, or swim bladder tumors in fish initiated as fry with N-methyl-N'-nitro-nitrosoguanidine (MNNG). FB1 was not a complete carcinogen in trout. No tumors were observed in any tissue of fish fed diets containing 0, 3.2, 23, or 104 ppm FB1 for a total of 34 weeks (4 weeks FB1 exposure, 2 weeks outgrowth on control diet, followed by 30 weeks FB1 diet) in the absence of a known initiator. FB1 promoted AFB1 initiated liver tumors in fish fed > or = 23 ppm FB1 for 42 weeks. A 1-week pretreatment of FB1 did not alter the amount of liver [3H]AFB1 DNA adducts, which suggests that short-term exposure to FB1 will not alter phase I or phase II metabolism of AFB1. In MNNG-initiated fish, liver tumors were promoted in the 104 ppm FB1 treatment (42 weeks), but FB1 did not promote tumors in any other tissue. Tumor incidence decreased in kidney and stomach in the 104 ppm FB1 treatment of MNNG-initiated trout. The FB1 promotional activity in AFB1-initiated fish was correlated with disruption of sphingolipid metabolism, suggesting that alterations in associated sphingolipid signaling pathways are potentially responsible for the promotional activity of FB1 in AFB1-initiated fish.

Treatment of aged rat sensory neurons in short-term, serum-free culture with nerve growth factor reverses the effect of aging on neurite outgrowth, calcium currents, and neuronal survival.

Impaired NGF production and release has been documented in aged animals, suggesting that decreased NGF receptor stimulation may be one factor contributing to neuronal dysfunction with aging. Other studies have suggested that aging may be associated with impaired intracellular responses to NGF. Because aging-associated neuronal dysfunction contributes to morbidity and mortality in the geriatric population, it is important to determine whether the effects of aging on sensory neuron function and survival are reversible. In the present study, we observed significantly decreased neurite outgrowth and neuronal survival in short-term cultures (0-96 h) of dorsal root ganglion (DRG) neurons from aged (>22 months) Fisher 344 x Brown Norway F1 hybrid rats, compared to young (4-6 month) and middle-aged (14 month) animals. From 24 to 96 h in culture, diminished survival of aged neurons appeared to be due to an increased rate of apoptotic cell death. DRG neurons from aged animals also exhibited significantly decreased whole cell, high-threshold voltage-dependent calcium currents, with a larger proportion of L-type current, compared to youthful and middle-aged animals. Treatment of aged DRG neurons with NGF restored neurite outgrowth, neuronal survival and calcium current amplitude and subtype distribution to those observed in youthful DRG neurons.

Neoplasia in zebrafish (Danio rerio) treated with 7,12-dimethylbenz[a]anthracene by two exposure routes at different developmental stages.

Using zebrafish, Danio rerio, initial pioneering work in the 1960s revealed carcinogen responsiveness of fish, yet very few subsequent tumorigenesis investigations have utilized this species. We exposed embryos (60 hours postfertilization) and fry (3 week posthatch) to 7,12-dimethylbenz[a]anthracene (DMBA) by immersion in aqueous solutions for 24 hours, at concentrations of 0-1 or 0-5 ppm (mg/L), respectively. Juvenile zebrafish 2 months posthatch were fed a diet containing 0-1,000 ppm DMBA for 4 months. Fish were sampled for histologic evaluation at 7-12 months after the onset of carcinogen treatment. Fry were most responsive to DMBA and showed the widest diversity of target tissues and histologic types of neoplasia, having several types of epithelial, mesenchymal, and neural neoplasia. The principal target tissues for carcinogenic response were liver following embryo or fry exposure, with gill and blood vessel the second and third most responsive tissues in fry. Intestine was the primary target and gill a secondary target in fish that received dietary DMBA as juveniles. These studies indicate that young zebrafish are most responsive to DMBA, showing a greater diversity of neoplasm types than rainbow trout. Thus, zebrafish are a valuable model system in which to study mechanistic aspects of the carcinogenesis process.

Neoplasia in zebrafish (Danio rerio) treated with N-methyl-N'-nitro-N-nitrosoguanidine by three exposure routes at different developmental stages.

We exposed embryos (83 hours postfertilizaton) and fry (3 weeks posthatch) to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) by immersion in aqueous solutions of 0-10 ppm for 1 hour (embryo) or 0-2 ppm for 24 hours (fry). Zebrafish embryos were microinjected with MNNG at levels of 0 or 96 ng/egg. Diets containing 0-2,000 ppm MNNG were fed to juvenile zebrafish for 3 months beginning at 2 months posthatch. Fish were sampled for histopathologic study at 6-12 months after initiation of carcinogen exposure. Embryos and fry were both quite responsive to MNNG; however, juvenile zebrafish were remarkably refractory to MNNG-induced neoplasia. Principal target organs in zebrafish treated as embryos with MNNG were liver and testis, with hepatocellular adenoma the most prevalent hepatic neoplasm. A variety of mesenchymal neoplasms occurred in zebrafish following embryo exposure to MNNG, including chondroma, hemangioma, hemangiosarcoma, leiomyosarcoma, and rhabdomyosarcoma. Testis and blood vessels were primary target organs for MNNG following fry exposure, with seminoma, hemangioma, hemangiosarcoma, and various other epithelial and mesenchymal neoplasms occurring. The zebrafish is a responsive, cost-effective lower vertebrate model system in which to study mechanisms of carcinogenesis.

Hemodynamic dysfunction and cytochrome P4501A mRNA expression induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin during embryonic stages of lake trout development.

Lake trout embryos exposed to [(3)H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) manifest toxicity after hatching by subcutaneous edema of the yolk sac, pericardial edema, meningeal edema, subcutaneous hemorrhages, and a marked congestion of blood flow in various vascular beds culminating in death. Our objective was to determine if this syndrome was associated temporally with morphologic lesions in the vascular endothelium, increased vascular permeability, and cytochrome P4501A (CYP1A) mRNA induction. Lake trout embryos exposed as fertilized eggs to TCDD were found to exhibit marked reductions in perfusion of the peripheral vasculature during the early sac fry stage of development (stage F(1)9), which consistently preceded other gross lesions and mortality observed later in sac fry development (stage F(2)10). This reduction in blood flow was manifested as severe capillary congestion and hemoconcentration in certain vascular beds. Transmission electron microscopic (TEM) examination of endothelial cells in these vascular beds failed to reveal cellular necrosis at hatching (stage E(5)8) and throughout sac fry development (stages F(1)9-F(2)10). Rather, only subtle ultrastructural changes in endothelial cells were found consisting of increased vacuolation, separation of intercellular junctions, and cytoplasmic blebbing, consistent with the TCDD dose and time course for developmental cardiovascular toxicity, which began to manifest itself in some embryos approximately 1 week prior to hatching (E(5)8). To assess permeability of yolk sac vasculature to certain constituents in blood, sac fry (stage F(2)10) were analyzed for the presence of plasma proteins, granulocytes, and serum creatine kinase activity in yolk sac subcutaneous edema fluid from control and TCDD-exposed treatment groups. TCDD dose- and time-related increases in yolk sac edema volume, plasma protein content of edema fluid, granulocyte concentration, and creatine kinase activity in the fluid were observed in midstage and late stage of sac fry development (stage F(2)10). Thus, yolk sac subcutaneous edema fluid is an ultrafiltrate of blood and results from increased vascular permeability. In contrast to the changes in vascular blood flow and permeability induced by TCDD during stages F(1)9 and F(2)10 of sac fry development, respectively, CYP1A mRNA levels were induced by TCDD as early as the 10-somite embryo (stage E(2)5). TCDD also caused a dose-related increase in CYP1A mRNA levels in sac fry at hatching (stage E(5)8) and throughout sac fry development (stages F(1)9-F(2)10). We conclude that subtle, ultrastructural changes in vascular endothelial cells consistently precede increases in vascular permeability and sac fry mortality; however, induction of CYP1A mRNA occurs prior to any observable morphological lesions, changes in vascular permeability, or sac fry mortality.

Altered NGF regulation may link a genetic predisposition for hypertension with hyperactive voiding.

PURPOSE: Hyperactive voiding and elevated smooth muscle NGF output are traits of the spontaneously hypertensive rat (SHR). Elevated target-derived NGF is associated with hypertension and hyperactive voiding in SHRs. In the present study, we tested for possible genetic links between hypertension, hyperactive voiding and augmented bladder smooth muscle cell (BSMC) NGF secretion. MATERIALS AND METHODS: We crossed SHRs with WKYs to produce a gene segregating F2 population. We measured F2 mean arterial blood pressure (BP) and six-hour voiding frequency. BSMCs were cultured from 'Low BP F2s' (95+/-2) and 'High BP F2s' (141+/-3 mm. Hg) and conditioned medium tested for NGF with a two-site ELISA. The NGF regulators isoproterenol, platelet-derived growth factor (PDGF) and phorbol-12-myristate-13-acetate were tested in F2 BSMC cultures. RESULTS: A positive correlation (r = 0.75) between blood pressure and voiding frequency existed in this F2 population. As BP rose voiding frequency increased and volume per void decreased such that there were no significant changes in total urine voided (Low BP F2s: 1.0+/-0.5; High BP F2s: 6.2+/-0.5 voids/6 hours). Low BP F2s (2.0+/-0.2) secreted NGF at a higher basal rate than High BP F2s (0.7+/-0.1 fg NGF/hr/100 cells). However, High BP F2s (1,620 and 3,850) were oversensitive to isoproterenol and PDGF-induced increases in NGF output, compared with Low BP F2s (219 and 1,282% control, respectively). CONCLUSIONS: Elevated tissue NGF due to a hypersensitivity to NGF regulating stimuli, rather than alterations in basal NGF, may genetically link hypertension and hyperactive voiding.

2,3,7,8-Tetrachlorodibenzo-p-dioxin alters cardiovascular and craniofacial development and function in sac fry of rainbow trout (Oncorhynchus mykiss).

Hallmark signs of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity in rainbow trout sac fry, are yolk sac edema, hemorrhage, craniofacial malformation, and growth retardation culminating in mortality. Our objective was to determine the role of cardiovascular dysfunction in the development of this toxicity. An embryotoxic TCDD dose (385 pg/g egg) caused a progressive reduction in blood flow in rainbow trout sac fry manifested first and most dramatically in the 1st and 2nd branchial arches and vessels perfusing the lower jaw. Blood flow was reduced later in the infraorbital artery and occipital vein of the head as well as segmental vessels and caudal vein of the trunk. Reduced perfusion occurred last in gill branchial arteries involved with oxygen uptake and the subintestinal vein and vitelline vein involved with nutrient uptake. Although heart rate throughout sac fry development was not affected, heart size at 50 days post-fertilization (dpf) was reduced far more than body weight or length, suggesting that the progressive circulatory failure caused by TCDD is associated with reduced cardiac output. Craniofacial development was arrested near hatch, giving rise to craniofacial malformations in which the jaws and anterior nasal structures were underdeveloped. Unlike the medaka embryo, in which TCDD causes apoptosis in the medial yolk vein, endothelial cell death was not observed in rainbow trout sac fry. These findings suggest a primary role for arrested heart development and reduced perfusion of tissues with blood in the early-life stage toxicity of TCDD in trout.

Experimental hepatic tumorigenicity by environmental hydrocarbon dibenzo[a,l]pyrene.

There is an evident need of low-cost vertebrates to be used in experimental carcinogenesis. Medaka (Oryzias latipes) provide a useful vertebrate model system for investigating tissue tropism of carcinogens and the action mechanisms of environmental contaminants posing a potential risk to human health. Juvenile medaka 2 months of age fed diets containing 100 ppm (dry weight basis) dibenzo[a,l]pyrene (DBP) for 28 days responded with hepatic neoplasia predominately of hepatocellular origin. When sampled 9 months following the termination of carcinogen exposure, medaka showed 26% incidence of neoplasia and 25% hepatic neoplasia, compared with 8% total neoplasia and 0% hepatic neoplasia in control fish. The predominant spontaneous neoplasms in this group of medaka were ovarian germ cell tumors. Hepatic neoplasia occurred at a higher incidence in female DBP-treated medaka than in males (11/29 vs 5/36). Nonneoplastic lesions observed in the livers of DBP-exposed fish included spongiosis hepatis, globular hyaline eosinophilic cytoplasmic inclusions in hepatocytes, foci of hepatocellular degeneration, extensive cytomegaly, and karyomegaly of hepatocytes. No activating exon I mutations in the one ras protooncogene examined were detected among six liver neoplasms. These results indicate that medaka are sensitive to the tumorigenic effects of the environmental carcinogen DBP, administered by dietary exposure.

Spinal and peripheral mechanisms contributing to hyperactive voiding in spontaneously hypertensive rats.

The influence of noradrenergic mechanisms involved in micturition in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats was investigated using continuous cystometry in in vivo and in vitro studies on isolated bladder and urethral tissues. Compared with WKY rats, SHR had a significantly lower bladder capacity (SHR: 0.7 +/- 0. 05 ml; WKY rats: 1.3 +/- 0.06 ml; P < 0.001), micturition volume (SHR: 0.4 +/- 0.04 ml, WKY rats: 1.2 +/- 0.05 ml; P < 0.001), and an increased amplitude of nonvoiding (unstable) bladder contractions. The effects of intrathecal and intra-arterial doxazosin on cystometric parameters were more pronounced in SHR than in WKY rats. There was a marked reduction in nonvoiding contractions after intrathecal (but not intra-arterial) doxazosin in SHR. Norepinephrine (0.1 microM-1 mM) failed to evoke contractions in bladder strips from WKY rats, in contrast to a weak contractile response in SHR. The response to electrical field stimulation was significantly less in bladder strips from SHR than from WKY rats. In WKY rats, norepinephrine produced concentration-dependent inhibition (87 +/- 5%, n = 6) of nerve-evoked bladder contractions. Almost no inhibition (11 +/- 8%, n = 6) was found in SHR. Alterations in bladder function of SHR appear to be associated with changes in the noradrenergic control of the micturition reflex, in addition to an increased smooth muscle and decreased neuronal responsiveness to norepinephrine. The marked reduction in nonvoiding contractions after intrathecal doxazosin suggests that the bladder hyperactivity in SHR has at least part of its origin in supraspinal and/or spinal structures.