Correlation of VEGF expression by leukocytes with the growth and regression of blood vessels in the rat cornea.
PURPOSE: To determine the temporal and spatial relationships between neovascularization and basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) mRNA and protein expression in the rat cornea after cautery with silver nitrate. METHODS: In female Sprague-Dawley rats, a silver nitrate applicator was placed on the central cornea to elicit circumferential angiogenesis, and blood vessel growth was quantified by digital image analysis of corneal flat-mounts. Total RNA or protein was extracted from whole corneas until 1 week after cautery, and bFGF and VEGF mRNA and protein levels were determined by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). To localize VEGF mRNA and protein, paraformaldehyde-fixed and paraffin-embedded histologic cross sections of corneas were examined by in situ hybridization and immunohistochemistry. Macrophages were identified by ED2 immunohistochemistry. To examine the regulation of VEGF, rats were treated with dexamethasone (0.5 mg/kg per day) and hyperoxia (70% O2). RESULTS: The neovascular response progresses in three phases: (1) a nonproliferative phase preceding vessel growth (< or = 48 hours after cautery); (2) a proliferative phase with maximal growth rate between 3 and 4 days; and (3) a regressive phase (day 7) with a decrease in vessel density accompanying the completion of vessel elongation. In corneas after cautery, bFGF mRNA expression was unchanged, and bFGF protein concentration decreaseed by 97% after 24 hours and returned to control levels by day 7. In contrast, VEGF164 and VEGF188 mRNA splice variants and protein peaked 48 hours after cautery, remained elevated 4 days after cautery, and decreased to near baseline by day 7. The peak concentration of VEGF in the cornea at 48 hours was calculated to be 720 pM, which is sufficient to evoke a functional response. In situ hybridization and immunohistochemistry showed VEGF expressed initially in neutrophils (24 - 48 hours) and subsequently in macrophages (4 days) adjacent to the cautery site. Treatment with either dexamethasone or systemic hyperoxia inhibited both neovascularization and the increase in VEGF expression. Dexamethasone inhibited 27% of cautery-induced VEGF upregulation at 24 hours and 23% at 48 hours, hyperoxia inhibited 32% at 24 hours and 43% at 48 hours, and combined treatment with both dexamethasone and hyperoxia had an additive effect (56% inhibition at 24 hours). CONCLUSIONS: VEGF production by leukocytes correlates temporally and spatially with cautery-induced angiogenesis in the rat cornea. Both inflammatory products and hypoxia appear to sufficiently increase VEGF expression near the cautery lesion to increase vascular permeability of limbal vessels and induce endothelial cell migration and proliferation. (+info)
Non-functioning pituitary tumour after long-term treatment with gonadotrophin-releasing hormone agonists in a patient with vaginal agenesis who underwent neovaginoplasty and cauterization of endometriosis under laparoscopy.
Vaginal agenesis combined with a functional uterus is a rare condition in which treatment modalities that preserve reproductive function are controversial. A 21 year old female presented with congenital vaginal agenesis combined with cervical atresia. She was treated with gonadotrophin-releasing hormone (GnRH) agonists for a total period of over 5 years when a non-functioning pituitary tumour was detected by brain magnetic resonance imaging (MRI). A laparoscopically assisted reconstruction of a neovagina and neoendocervical canal was performed utilizing lyophilized porcine dermal skin to line the neovagina. Endometriosis of the pelvis was revealed and adhesiolysis and cauterization were also carried out under laparoscopy. The GnRH agonist was discontinued and the patient resumed cyclic menses with no abdominal pain. The pituitary tumour decreased in size 6 months after the cessation of GnRH agonists. We raise the question as to whether pituitary MRI should be performed for patients who need long-term administration of GnRH agonists. (+info)
Role of the vomeronasal organ in neonatal offspring recognition in sheep.
Twenty-five pregnant Dorsett ewes were randomly divided into three groups to test if ewes use their vomeronasal organs for offspring recognition during nursing. One group of eight ewes (procaine) were made anosmic by irrigation of the nasal olfactory apparatus with a zinc sulphate procaine solution. The second group of nine ewes (cauterized) had their vomeronasal organs rendered nonfunctional by cauterization of the nasoincisive duct. The third group of eight ewes were the controls. Parturition was synchronized in all ewes with betamethasone on Day 145 of gestation. Maternal responsiveness was tested two separate times with 1- to 2-day-old alien lambs. Each alien lamb trial was conducted 24 h apart. Cauterized ewes allowed alien lambs to suckle and they were unable to distinguish alien lambs from their own lambs, whereas the ewes in both groups with functional vomeronasal organs (procaine and control) violently rejected any alien lamb's attempt to suckle. Thus, female sheep use their vomeronasal organs for neonatal offspring recognition. (+info)
An evaluation of the role of leukocytes in the pathogenesis of experimentally induced corneal vascularization.
Studies of corneal explants in the hamster cheek pouch chamber have demonstrated that blood vessels invade the cornea only if the tissue is first infiltrated by leukocytes. In view of this observation, a comparative study of the events that precede and accompany corneal vascularization was undertaken in various experimental models. A variety of established methods were used to induce corneal vascularization, including exposure of the cornea to noxious agents, intracorneal injection of antigens into sensitized animals, as well as maintaining animals on diets deficient in vitamin A or riboflavin. In all models studied, the corneal vascularization was a manifestation of the reparative phase of the inflammatory response. A conspicuous leukocytic infiltrate of the cornea preceded and accompanied the corneal vascularization in all of the models. Although the lesions varied in several respects in the different models, all models displayed three phases with regard to vascularization: an early prevascular phase of leukocytic infiltration, a second phase where blood vessels persisted in the cornea in the absence of leukocytes. The latent period that preceded vascularization was directly related to the time of the initial leukocytic infiltration. The models in which a delay occurred in the leukocytic invasion displayed a subsequent delay in the vascular ingrowth. Conversely, in experiments where there was a rapid and extensive leukocytic invasion, there was also an early and enhanced corneal vasoproliferative response. In the various modesl investigated, the sites of the leukocytic infiltration and subsequent vascular ingrowth into the cornea paralleled each other. The data further support the hypotheses that leukocytes are a prerequisite to corneal vascularization and that leukocytes produce one or more factors which stimulate directional vascular growth. (+info)
Adult deficiencies and duplications of head and thoracic structures resulting from microcautery of blastoderm stage Drosophila embryos.
Drosophila embryos were damaged by microcautery at the cellular blastoderm stage at the sites of presumptive imaginal cells, identified from fate maps. The resulting adults were analyzed for abnormal structures. Cautery of any of the presumptive imaginal regions can lead to defects in the adult cuticle, though the majority of adults which hatch are morphologically normal. The abnormal adults had one or more discs either deleted, incompletely formed, or misarranged. Several of the structures which were incomplete had duplicated regions. The results suggest that, from the time fo their initial formation in the cellular blastoderm, a group of cells determined to be an 'adult' structure possesses a gradient of developmental capacity which is expressed by certain regions duplicating and other cells regenerating. The types of duplications found were similar to those resulting from other experimental treatments of imaginal discs at later stages in development, indicating that the presumptive imaginal disc cells, when they are first established in the cellular blastoderm, have a similar organization to mature imaginal discs. (+info)
Effects of ovarian follicle ablation on FSH, oestradiol and inhibin A concentrations and growth of other follicles in sheep.
The aim of this study was to examine the effect of removal of the largest follicle or all visible follicles during the first follicle wave on subsequent follicular growth, steroid, inhibin A and gonadotrophin secretion in sheep. On day 4.5 of a synchronized oestrous cycle, ewes (n = 18) were assigned to one of three groups which underwent either no treatment (control), ablation of the largest follicle (largest follicle aspirated and cauterized via laparotomy) or ablation of all follicles (all visible follicles ablated). Between day 0 and day 10 of the oestrous cycle, blood samples were collected every 8 h and ovaries were examined daily using transrectal ultrasonography. The lifespan of the second largest follicle (number of days > 3 mm in diameter) was longer (6.7 +/- 0.9 days; P < 0.05) and the maximum diameter tended to be greater (4.8 +/- 0.3 mm; P = 0.07) in ewes in which the largest follicle was ablated than in the control ewes (3.8 +/- 0.4 days; 4.2 +/- 0.3 mm). There was no difference in the day of emergence of the second follicular wave between groups (day 6.9 +/- 0.4). However, the peak of the transient increase in FSH concentrations after ablation was earlier (day 5.67 +/- 0.15; P < 0.05) in ewes in which all follicles were ablated than in control ewes (day 6.72 +/- 0.36); the timing in ewes that had only the largest follicle ablated was intermediate (day 6.11 +/- 0.28). Serum inhibin A concentrations were about three-fold lower (P < 0.05) in both follicle ablation groups than in the control group. The numbers of follicles 2-3 mm in diameter during the first 3 days of the second follicular wave were greater in 'ablated ewes' (both groups had 2.6 +/- 0.2 follicles day-1) than in control ewes (1.7 +/- 0.3 follicles day-1). It is concluded that: (i) transient increases in FSH concentrations precede the emergence of follicle waves; (ii) ablation of all follicles on day 4.5 after oestrus advanced the timing of the next peak in FSH concentrations and the numbers of small follicles associated with the development of the second follicular wave; and (iii) ablation of the largest follicle resulted in an increase in the lifespan of the second largest follicle, indicating a regulatory role of large dominant follicles over smaller subordinate follicles. (+info)
Histological study on the influences of an ultrasonic scalpel on skeletonized vessel wall.
OBJECTIVE: The objective of this study was to histologically clarify the difference of vascular wall damage when an ultrasonic scalpel is used in varied ways in the vicinity of a vessel. SUBJECTS AND METHODS: 1) The surface of sodium carbonate-containing jelly was manually brushed with the edge of a dissecting hook type Harmonic Scalpel (HS), and the thickness of the air bubble layer was measured to investigate the range to which the vibrations of the instrument reached. 2) The internal thoracic artery (ITA), radial artery (RA) and vein skeletonized were cut bluntly or brushed using HS ex vivo, and tissue damages were observed histologically. 3) The depth of thermal degeneration (TD) of residual stumps of ITAs skeletonized by HS using an output power level (level) of 2 and the quick touch method at the time of coronary arterial bypass grafting (CABG) were investigated histologically. RESULTS: 1) The mean thickness of the air bubble layers by single brushing was 3.7, 3.7 and 3.1 mm at level 4, 3 and 2, and no significant difference. When brushed 5 times, it was 6.9, 5.5 and 6.7 mm, respectively, showing marked increases compared with single brushing. 2) A: One side of the RA stump cut with a dissecting hook at level 2 was nicely occluded by a degenerated protein coagulum, but the contralateral had no coagulum. An ITA cut by a shear type blade at level 3 showed that both stumps were nicely occluded, but the vessel wall was introverted and fragmented. B: ITAs brushed 5 or 10 times at level 2 showed that TD occurred in tunica externa, the mean depth of 100 or 203 microm, and never exceeded the external elastic lamella. RAs brushed 10 times at level 2 and 3 showed that TD and air bubble generation occurred in the tunica externa, and the mean depth was 203 and 203 microm. However, TD exceeded the external lamella in some cases at level 3. Veins brushed 10 times at level 3 showed that TD spread to all layers. 3) The depth of TD in ITAs skeletonized clinically by HS was 400 to 530 microm, and apart from the external elastic lamella. CONCLUSIONS: 1) Though the air bubble layer was very thick in jelly, it was observed only in tunica externa ex vivo. 2) For coagulation and cut of small blood vessels, it is vital to press an HS blade edge onto the vessel so as to press equally both portions to be cut. There is a possibility of a fragmented and introverted vessel wall into the lumen. 3) By dissecting ITA and RA using HS at level 2 and the quick touch method, TD can be limited to the depth of the connective tissue of tunica externa. (+info)
The histological features of microwave coagulation therapy: an assessment of a new applicator design.
Microwave ablation of tumours within the liver may become an adjunct or alternative to resection in patients with primary or secondary cancers. This technique combines the benefits of a large, localized coagulative effect with a single insertion of the applicator, in a significantly shorter time than comparable treatments. A new range of microwave applicators were developed and tested in animal models and both ex-vivo and in-vivo specimens of human liver at resection. At laparotomy, the applicator tip was inserted into normal liver parenchyma and tumours, with each specimen subjected to irradiation for 180 s or more and at varying power outputs. On sectioning an area of spherical blanching was observed around the applicator cavity. Microscopically a zone of coagulative necrosis was seen adjacent to the site of probe insertion. Damage to blood vessels and bile ducts occurred distal to the probe cavity suggesting the passage of heated fluid, a finding that was diminished by temporary occlusion of the hepatic vasculature (a Pringle manoeuvre). Ultra-structural damage was confirmed within the burn zone and selected liver enzymes were shown to be functioning beyond this region. We suggest this indicates the surrounding liver parenchyma is functioning normally and therefore the volume of microwave-induced damage is controllable. We are confident that the new applicator design will allow the effective treatment of larger tumours in a safe and controlled manner with a single application of energy. (+info)