Browsing by Author "Smith, Bonnie J."

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    Advanced Studies in Veterinary Anatomy: Angiogenesis in Caprine Reproductive Organs of Non-Pregnant and Pregnant Normal and Swainsonine-Treated Does
    Hafez, Shireen Abdelgawad (Virginia Tech, 2005-04-20)
    The female reproductive organs are among the few adult tissues in which periodic angiogenesis normally occurs. Pathological angiogenesis can occur in various conditions, such as solid tumors. Vascular endothelial growth factor (VEGF) signaling often represents a critical rate-limiting step in physiological and pathological angiogenesis. This study utilizes development of utero-ovarian vasculature during pregnancy in goats as a model of physiological angiogenesis. Non-pregnant does and does at 4, 7, 10, 13, 16, and 18 weeks of gestation were used. Arteries of the reproductive tract were injected in situ with Microfil®. The tracts were fixed, dehydrated, and rendered transparent to reveal the paths of arteries. The ovarian artery was tortuous and lay in close apposition to the uterine tributary of the ovarian vein in all specimens studied. In non-pregnant does, this arrangement may serve as a local utero-ovarian pathway for the corpus luteum (CL) luteolysis at the end of non-fertile estrous cycle. During pregnancy, this arterio-venous arrangement may transfer luteotropic substances from uterus to ovary, which may serve in maternal recognition of pregnancy and fit the fact that the goat is CL dependent throughout gestation. In some cases of triplets, the size of the uterine branch of the ovarian artery was equal to or even larger than that of its parent artery and/or the ipsilateral uterine artery; and the vaginal artery contributed a connecting branch to the uterine artery. These physiological adaptations of the ovarian and/or vaginal arteries correlate well with the increasing nutrient demands of the growing multiple fetuses. In a second experiment, the vasculature of the uterus and ovaries was injected in situ with a mixture of Batson's No.17® and methyl methacrylate and then processed for observation by SEM. The microvasculature differed between non-pregnant and pregnant does, and with advancing gestation. We concluded that goats possess a multivillous type placenta. Capillary sinusoids and crypts on the fetal surface of the caruncle may compensate for the negative effect of the increased interhemal distance. Intussusceptive angiogenesis should be considered as equally possible and important mechanism as sprouting angiogenesis during placental development. Capillary diameters increased significantly during pregnancy especially after 4 weeks. Capillary density index was 66.8, 68.7, 55.5, 63.5, 70.1, 70.4, 64.5 percent in non-pregnant, 4, 7, 10, 13, 16, and 18 weeks of pregnancy, respectively. In the ovary, coiling of the ovarian branch of the ovarian artery around the ovarian tributary of the ovarian vein was observed. This may represent a local channel required for product transport from ovarian vein to ovarian artery and might have a role in regulating blood pressure to various ovarian structures. Immunolocalization of VEGF was performed as a third experiment. Immunostaining was observed in cyto- trophoblasts, maternal epithelial tissues, and vascular endothelium and smooth muscle, but not in binucleate giant cells or connective tissue. No apparent differences were observed in intensity and pattern of VEGF staining associated with advancing gestation. Luteal and follicular cells, and endothelium and smooth muscles of the ovarian vasculature positively stained. Patterns and intensity of staining of VEGF suggest that the fetus is directing its own survival by producing growth factors affecting fetal and maternal tissues. VEGF may have a role in growth and differentiation of cytotrophoblasts, as well as, development and maintenance of CL. In the fourth experiment, the sequential expression of VEGF and its receptors (fms-like tyrosine kinase, Flt-1 and kinase-insert domain-containing receptor, KDR) was measured using real-time quantitative PCR. Targets were detected in all studied tissues; however, levels of expression differed according to the stage of pregnancy. Expression of VEGF and its receptor mRNAs increased with advancing pregnancy, which correlates with the expansion of vasculature during pregnancy. Differences in the time-courses of the expression of Flt-1 and KDR mRNAs during pregnancy suggest that each receptor plays a different role in the angiogenic process. As an application of our model of angiogenesis, we tested the effect of swainsonine (active compound of locoweed and a potential anti-cancer drug) on the process. Does treated with swainsonine were euthanized at 7 and 18 weeks. No significant differences were found in sinusoidal diameters in treated does at 7 weeks, but a decrease in capillary density index was noted. In the ovary, focal avascular areas were observed in the corpus luteum of swainsonine-treated does at 7 weeks of pregnancy. Swainsonine caused great distortion in the uterine and ovarian vasculature at 18 weeks. A decrease in intensity of the immunoreactivity to VEGF antibody was observed in tissues from swainsonine-treated does at 7 and 18 weeks. There was no substantial effect of swainsonine on the expression of VEGF and its receptors' mRNAs in any of the studied tissues (except in the left ovary, where it had an inhibitory effect) at 7 weeks of pregnancy, but it had an inhibitory effect at 18 weeks. Demonstration of swainsonine's potential to negatively affect vascular development and suppress genes likely involved in angiogenesis at critical stages of blood vessel proliferation lends credibility to its potential as anti-cancer drug.
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    The Effects Of Mercuric Chloride On Cultured Atlantic Spotted Dolphin (Stenella Plagiodon) Renal Cells And The Role Of Selenium In Protection
    Wang, Amy (Hui-Shan) (Virginia Tech, 1998-04-24)
    Marine mammals are known for their low susceptibility to mercury toxicity, and it was hypothesized that selenium may play a role in protection against mercury toxicity. To gain insight into the mechanisms of the low susceptibility of cetaceans, we investigated the in vitro effects (1) of mercuric chloride (HgCl₂) on the ultrastructure and cell death of Atlantic spotted dolphin renal cells (Sp1K cells), (2) of HgCl₂ on the cell proliferation and cell cycle status of Sp1K and Rhesus monkey renal cells (MK2), and (3) of sodium selenite (Na₂SeO₃) on cell proliferation and cell death of control and HgCl₂-treated Sp1K cells. HgCl₂ affected multiple organelles and nuclei in Sp1K cells, and induced apoptosis in a time-and dose-dependent manner. Both ultrastructural changes and induction of apoptosis were milder than seen in other cell types in previous publications. In addition, Sp1K cells were able to proliferate at 25 µM HgCl₂ while MK2 cells were killed at 15 µM HgCl₂. An increase in percentage of cells in the G0/G1 phase in the cell cycle and a decrease in S, and G2/M phase cells were seen in Sp1K cells exposed to more than 10 uM HgCl₂ more than 72 hours. MK2 cells showed cell cycle changes only at 24 hours exposure, and may be due to a sensitive subgroup. These data suggested that Sp1K cells were less susceptible than other cell types in a cell-specific way, which was independent of selenium protection. Concurrent exposure to Na₂SeO₃ provided protection against the HgCl₂-induced decrease in cell proliferation of Sp1K. The protective effects were greater if Na₂SeO₃ and HgCl₂ were premixed, but disappeared if exposures did not overlap. Although pretreatments with Na₂SeO₃ alone did not provide protection, they increased the protection of selenium administered later. Furthermore, Na₂SeO₃ decreased HgCl₂-induced apoptosis. These data demonstrated the Na₂SeO₃ protection against HgCl₂ toxicity in Sp1K cells in terms of cell proliferation and apoptosis. This study is the first report that reveals the existence of mercury-selenium antagonism in cultured cetacean cells. The data supported the hypothesis that selenium protection against mercury toxicity is, at least partially, through competition of binding sites and formation of mercury-selenium complex.
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    Exposure to formaldehyde at therapeutic levels decreases peripheral blood lymphocytes and hematopoietic progenitors in the pronephros of tilapia Oreochromis niloticus
    Holladay, Steven D.; Smith, Bonnie J.; Gogal, Robert M. (Inter-Research, 2010)
    Formaldehyde (HCHO) was recently detected at concentrations above the cancer benchmark in 90% of 60 000 surveyed United States census tracks. Formaldehyde leaches into and mixes with water extremely well, exposing aquatic life. Further, formaldehyde is used therapeutically in aquaculture to remove external protozoa and other parasites from fish. The present study was undertaken to determine if sub-acute HCHO causes immunologic changes in tilapia Oreochromis niloticus. Fish were exposed to 0, 50, or 150 ppm of HCHO. Immune parameters examined included blood hematology, spleen/body weight ratio, spleen and pronephros total cellularity, leukocyte blastogenesis, and natural killer cell cytotoxicity. Organ/body weight ratios and total cellularity were not different from controls. Similarly, mitogen response and natural killer cell function were unchanged. Peripheral blood lymphocytes decreased as HCHO exposure increased. Formaldehyde exposure also decreased the number of progenitor cells in the fish pronephros. These observations suggest possible immunosuppressive effects of HCHO in fish.
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    Gross and Microscopic Observations on the Lingual Structure of the West Indian Manatee (Trichechus manatus latirostris)
    Levin, Milton Jay (Virginia Tech, 2004-03-19)
    The West Indian manatee tongue was examined macroscopically, light microscopically, and electron microscopically (scanning and transmission). The tongue was slender, muscular, and firmly fixed in the oral cavity. Only the cranial tip was free and mobile. Numerous filiform papillae were distributed over the dorsal surface of the rostral lingual region. Caudal to the filiform papillae, multiple raised, round papillae were distributed over the majority of the dorsum. Fungiform papillae were restricted to the lateral margins of the tongue. Caudally, the dorsal and lateral regions showed numerous open fossae and pits. Microscopic examination showed the majority of the lingual dorsum to be covered with a thick stratified squamous epithelium. The caudal dorsal and lateral open pits led to well-developed mucous salivary glands. Foliate papillae, located on the caudal region of the tongue, contained taste buds embedded in the epidermis. Glands within the foliate papillae were mostly mucous, though some seromucous glands were evident. Throughout the tongue, striated muscle was abundant below the epidermis. Blood vessels, lymph channels, and nerve fibers were freely distributed throughout the intermuscular stroma. Nerve fibers reacted positively with neuron specific enolase antibody throughout the lingual structure, including nerve bundles, muscle bundles, glands, and taste buds. Electron microscopy revealed cytoplasmic vacuoles juxtaposed to the nucleus in the stratum spinosum of the foliate papillary region.
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    Immune Function Determination in Mice Dermally Exposed to Permethrin
    Punareewattana, Korawuth (Virginia Tech, 1998-03-30)
    Inhibited immune responses have been observed following occupational, inadvertent, or therapeutic exposure to chemically diverse xenobiotics. In the present studies, preliminary data were generated showing limited but significant systemic immunotoxicity following low-level topical exposure to the pyrethroid insecticide, permethrin (formerly not considered an immunotoxicant). Permethrin was applied to the shaved dorsal interscapular region of C57Bl/6N mice at doses of 0.5, 1.5, or 5.0 μl/day. The highest of these doses was approximately equal to 215 μg/kg/day, which is about seven times the estimated daily human exposure in individuals wearing permethrin treated clothing for insect protection. Mice were thus exposed to permethrin daily for 10 or 30 consecutive days, or every other day for 7 or 14 exposures. Body weight was not affected by the treatment. However thymic weight was decreased and splenic weight increased 2 days after termination of the topical exposure. Histopathology of immune organs showed no significant changes. Splenic macrophages showed significantly depressed chemiluminescent responses up to 10 days following termination of exposure, but macrophage phagocytic activity was not affected. Cell surface markers of thymocytes, splenocytes and bone marrow cells were not affected. Antibody production as shown by plaque forming cell (PFC) assay decreased significantly at 10 days after dosing termination. Taken together, these data indicate that low-level topical permethrin exposure may produce systemic immunotoxicity.
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    Immunotoxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and Diethylstilbestrol (DES) in the Fetal Mouse Thymus and Liver
    Besteman, Elizabeth Gayle (Virginia Tech, 2006-05-10)
    Diethylstilbestrol (DES) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have been identified as immunotoxicants causing thymic atrophy, thymocyte hypocellularity, phenotypic changes detected by CD4 and CD8 surface antigens, and progenitor T-cell targeting in the fetal mouse. We hypothesized that gestational exposure to these two compounds may lead to comparable histologic and gene expression alterations in the fetal mouse thymus and liver. Treatment of pregnant C57Bl/6 mice with doses of 5 or 10 ug/kg TCDD or 48 ug/kg DES by oral gavage on gestation days (gd) 14 and 16 severely depressed day 18 thymic cellularity. Histologic evaluation of day 18 fetal thymuses showed disruption of normal cortico-medullary architecture after TCDD or DES. Decreased thymocyte density was noted primarily in cortical zones where pyknotic cells were increased by either TCDD or DES treatment. Using day 18 thymocyte suspensions and flow cytometry, 7-AAD showed decreases in viable thymocytes from TCDD- or DES-treated fetal mice, and concomitant increases in thymocytes in early apoptosis. When thymocytes were co-identified with CD4 and CD8 cell surface antigen expression, enhanced apoptosis occurred in CD4+CD8+ phenotype after TCDD treatment. After DES exposure, increased apoptosis occurred in CD4-CD8- and CD4-CD8+thymocytes. Both TCDD and DES increased liver to body weight ratios and decreased ratios of hematopoietic to hepatic cells present. Cytomegaly was seen in hepatocytes of TCDD and DES treated animals, and these cells had more variable features, such as increased cytoplasmic basophilia and more prominent nucleoli. Real time quantitative PCR demonstrated that DES decreased c-jun, Bcl-2, and PKCalpha mRNA expression. These results suggest a shift away from proliferative activity and may reflect alterations noted predominantly in the hematopoietic population. TCDD increased c-jun mRNA expression with modest decreases in PKCalpha, and marked decreases in p53 also noted. Decreases in p53 suggest a pro-proliferative status of hepatic cells, while decreases in PKCalpha may indicate decreases in phosphorylation of substrates required for normal cell cycle progression. The increased c-jun suggests that this gene may play a role in the hepatocyte hyperplasia, as well as the diminution of hematopoiesis.
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    Improved Late-gestation Cardiac Morphology in Fetuses of Diabetic Mothers After Maternal Immune Stimulation: Potential Role of Dysregulated Apoptosis
    Gutierrez, Juan Claudio (Virginia Tech, 2009-01-22)
    The incidence of malformed newborns is higher in human pregnancies complicated by diabetes mellitus, as compared to non-diabetic pregnancies. Neural tube and cardiac defects predominate among the fetal malformations induced by hyperglycemia. Non-specific maternal immune stimulation is protective in mice against birth malformations caused by chemical or physical teratogens, or by maternal diabetes mellitus. Insulin dependent diabetes was induced in ICR females to study the late gestation fetal heart by morphometric analysis. Diabetic females treated with Freund's compete adjuvant (FCA) or interferon-gamma (IFNγ) were also generated to elucidate potential positive effects of maternal immune stimulation during the diabetic pregnancy by morphometric analysis and pathologic scoring. Insulin-dependent CD1 females were generated to analyze late gestation fetal myocardial apoptosis by flow cytometric analysis and by real time-polymerase chain reaction (RT-PCR) analysis of a panel of 5 genes involved in apoptosis/proliferation (Bcl-2, P53, Caspase3, Caspase9 and PkC-e). The morphometric analysis of fetal hearts revealed visibly obvious dilation of ventricular chambers and outflow channel of the left ventricle, and reduction of total myocardial ventricular area in late gestation fetuses, as predominant changes seen in the offspring of diabetic dams. Pathologic scoring revealed that maternal immune stimulation, particularly with FCA, in part alleviated fetal heart changes of cavitary dilation and myocardial reduction. Increased rate of apoptosis/necrosis in the fetal myocardium in late gestation during the diabetic pregnancy was evidenced by flow cytometric analysis. Particularly there was a significant increase in percentage of early apoptotic cells in the fetal myocardium detected by cell markers annexin V and propidium iodide. There was also a significant increase in percentage of late apoptotic/necrotic fetal myocardial cells in the diabetic group compared to the control group. These results suggest that maternal treatment with FCA may in part protect the heart from high hyperglycemia by reducing the number of myocardial cells undergoing apoptosis and necrosis. The RT-PCR analysis revealed subtle changes in gene expression for all the genes except Bcl-2. A paradoxical and dramatic up-regulation of this anti-apoptotic gene was observed in late gestation fetal myocardium from the insulin-dependent hyperglycemic groups. Possibly, this could be a mechanism to protect the fetal myocardial cell from the chronic exposure to a severe hyperglycemic insult and consequent apoptosis. In conclusion, maternal insulin-dependent diabetes caused morphological changes in the late gestation fetal heart. Such changes were in part related to dysregulation of myocardial apoptosis. Maternal immune stimulation with FCA improved fetal heart morphology, by a mechanism that may in part relate to normalizing fetal myocardial apoptosis.
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    Microanatomic structure of cetacean skin in the urogenital region
    Jones, Flynn Margaret (Virginia Tech, 1993-08-05)
    It was hypothesized that there may be microanatomic specializations in the urogenital slit and mammary region of cetaceans. There may be an integumentary-linked mechanism in these animals similar to that which causes the milk let-down response in terrestrial mammals. This hypothesis was tested on tissue samples from fourteen animals collected in a standardized array of fourteen samples from the urogenital area, and one each from the ventral aspect of the flipper and the mid-dorsal body wall for comparison. Using standard histological and ultrastructural procedures, including both transmission and scanning electron microscopy, tissues from nine species were investigated. These included the bottlenose dolphin (Tursiops truncatus), striped dolphin (Stenella coeruleoalba), harbor porpoise (Phocoena phocoena), pygmy sperm whale (Kogia breviceps), short and long finned pilot whales (Globicephala macrorhynchus and malaena respectively), beluga whale (Delphinapterus leucas), humpback whale (Megaptera novaeangliae), and fin whale (Balaenoptera physalus). Measurements were taken of the height of the epidermis, the thickness of the epidermal stratum externum, and the height and number of dermal papillae. Co1lagen bundles of the reticular dermis were measured and ranked by diameter. Nervous structures were quantitatively evaluated by type and location. No differences were found in the epithelial, connective, or nervous tissue of the skin in this region that would imply an increased sensitivity in this area. However, an observed unique organization of the connective tissue may imply a functional difference in the skin of the urogenital region unrelated to the milk let-down phenomenon. Possible alternative mechanisms for the initiation of milk let-down in cetaceans are discussed, including myoepithelial cell contraction caused by urogenital bumping by calves, vocalization by calvest and tacto/acoustic stimulation of the urogenital area by the calf. Epidermal thickness and papillary height varied among animals of different cetacean species, although there seemed to be a structural 'formula' applicable to the skin of all cetaceans that would permit efficient turnover of the epidermis. The significance of integumentary lipids is discussed. Deviations in cellular and subcellular structures of cetacean skin from those described in previous reports are mentioned, and a previously undescribed location for nerves in dermal papillae was revealed.
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    Ontogenic Morphology and Enzyme Activities of the Intestinal Tract of the Nile Tilapia, Oreochromis Niloticus
    Tengjaroenkul, Bundit (Virginia Tech, 2000-04-26)
    The gross intestinal configuration of the Nile tilapia intestine changed dramatically from a short, straight intestinal tube at hatch (day 0) to a very complex, coiling pattern first attained at 9 weeks post-hatch. During the developmental period, gut length increased from 90% to 410% of body length. The rate of increase in both intestinal and body lengths took place at an accelerating rate as the fish aged. The great intestinal length provides an advantage to the fish in digestion and absorption of nutrients present in the less energy-efficient herbivorous diet. Formulation of commercial diets to match the development of the fish's intestine may offer commercial advantage. Appearance, localization and distribution of intestinal enzymes were observed in the fish at hatch and at mature stages using enzyme histochemistry. At hatch (day 0), most gut enzymes were already present in the intestinal brush border. As the fish matured, activities of the enzymes were widely distributed along the intestinal tract. The early appearance and broad distribution of activities of all studied intestinal enzymes may be one factor contributing to the rapid growth rate characteristic of tilapia, which differs markedly from other fish species. To investigate the possibility of using alfalfa as a potential protein food replacement in tilapia, the effects of different levels of alfalfa in feeds on growth and intestinal enzyme activities were observed in the fish aged 3-15 weeks. Results demonstrated that replacing 20% and 40% of a commercial diet with alfalfa had an overall negative effect on body and intestinal growth, as well as the intestinal enzyme activities from age 3-9 weeks. Thus, using alfalfa as a food replacement is not optimal for fish of these young ages, but may yet be suitable for older fish.
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    The Roles of Age, Glomerular Location, and Collagen Expression in the Canine Kidney: Analysis of a Lifespan Study
    Pomeroy, Melinda J. (Virginia Tech, 2001-12-05)
    It is well documented that the incidence of renal disease, and therefore renal dysfunction, increases with age in many species of mammals. Such alterations in renal structure and function may significantly affect long-term toxicology studies. The purpose of this study was to assess the temporal evolution of glomerulosclerosis, an important renal lesion, in laboratory housed dogs, an important model system in chronic toxicological studies. We histopathologically examined representative sections of dog kidneys, quantified glomerular lesions (using the 0-5 scale of the World Health Organization classification system) and performed of statistical analysis of the extent and distribution of such changes. The kidney samples were obtained by necropsy, and occasionally biopsy, procedures from a collection of 159 purebred Beagle dogs maintained for their entire lifespan in well-controlled conditions. The lesions were correlated with sex, age, and intra-renal location of affected glomeruli to determine the relationship of each in the development of glomerulosclerosis. All dogs examined had some degree of glomerulosclerosis. In the youngest (up to 2 years of age), this was minimal, but was more advanced by middle age (3-7 years). The condition progressed with further aging and was associated with progressive fibrosis and tubular loss. Location and advancing age were significantly related to the development of glomerulosclerosis such that as age increases, the incidence of glomerulosclerosis increases, with the inner medullary ray and inner cortex demonstrating the highest occurrence. Using immunohistochemical analysis, the percentage of type IV collagen within glomeruli was determined. No significant increase in type IV collagen in glomeruli due to age or location was seen. An increase in type III or type V collagen within glomeruli was not apparent either, upon visual examination. This study indicates that renal lesions, including glomerulosclerosis, occur commonly and progress over the lifetime in a genetically similar population of laboratory Beagle dogs maintained under optimal standard environmental conditions. Such typical, age-related change needs to be taken into consideration when conducting chronic toxicological experiments using such animals.
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    Subacute immunotoxic effects of the environmental contaminants 7,12-dimethylbenzanthracene (DMBA), hexachlorocyclohexane (lindane), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on spleen and pronephros cellularity and morphology and functional activity of macrophages contained in these hemotopoietic organs in the cichlid fish tilapia (Oreochromis niloticus)
    Hart, Laura J. (Virginia Tech, 1996-07-15)
    Alterations of immune parameters were investigated in fish exposed to non-overtly toxic levels of three different environmentally relevant chemicals, 7,12-dimethylbenzanthracene (DMBA), hexachlorocyclohexane (lindane), and 2,3,7,8- tetrachlorodibenzo-p -dioxin (TCDD). Each chemical agent was administered to tilapia in separate experiments by intraperitoneal injection for five consecutive days. Following the final dose, total cellularity and histology of the spleen and pronephros were assessed, as were activity of phagocytic celis contained in these hematopoietic organs.

    Using chemical doses which produced no clinical toxicity, tilapia exposed to each chemical agent displayed a significant reduction in total cell number of both spleen and pronephros, in most cases in a dose-related manner. Consistent with this observation, splenic and pronephric hypocellularity was confirmed upon histological examination of chemical-treated fish. However, neither superoxide radical production or phagocytosis of splenic or pronephric macrophages was inhibited in either DMBA, lindane, or TCDD exposed fish. Results of this study indicate that depressed total cell number in fish hematopoietic organs may be a more sensitive indicator of exposure to these environmental contaminants than is the activity of macrophages contained within these organs.

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    Superficial temporal artery flap: its development and application in the dog and cat
    Fahie, Maria Aline (Virginia Tech, 1996-04-15)
    Cutaneous arterial blood supply to the temporal region was evaluated in 8 dogs and 8 cats. Dissection of 4 dog and 4 cat cadavers revealed the location of cutaneous branches of the superficial telnporaJ artery supplying the frontalis tnuscle and skin of the temporal region. The frontalis 1l1uscle is a thin muscle dorsal to the temporaJis muscle that extends cranially and rostral1y from the rostral border of the scutulU111 to the forehead and upper eyelid. Microangiography and subtraction radiography of the external carotid and superficial telnporal arteries were used in 4 dogs and 4 cats to determine arterial blood supply to the temporal region and frontalis muscle. A superficial temporal artery (ST A) flap was developed in 9 dogs [ group A (n=5), group B (n=4)]. Ligation of the superficial tetnporal artery in the control dogs (n=5), rendered flaps dependent on the subdermal plexus. Dogs in group A (n=5) and the control group (n=5) had flap lengths that extended to the contralateral eye, while group B (n=4) flaps extended to the contralateral zygomatic arch. A11 flap widths were equivalent to the width of the zygomatic arch in the individual dog. Mean length of surviving tissue (mean survival length) (+/- SD) of control flaps was 7.0 (0.6) ern, compared with experimental flaps, group A 9.) (0.8) em and group B 10.4 0.1) cm. Mean survival percentage area of control flaps was 73.5 (7.4) %, compared with experimental flaps, group A 93.1 (7.5) 0/0 and group B 69.1 (4.5) 0/0. The mean survival length of control and experimental flaps was significantly different (P < 0.05). There was no significant difference between survival lengths of the experimental groups.