Neuronal VEGF expression correlates with angiogenesis in postnatal developing rat brain.

When exposed to chronic sublethal hypoxia the developing brain responds with increases in permeability and angiogenesis. Vascular endothelial growth factor (VEGF) may mediate this response. Here, we present data on the localization of VEGF in the rat brain cortex during postnatal development and its correlation to vascularization. We reared newborn rats under normoxic conditions and in hypoxic chambers (FiO(2) 9.5%), removed them at postnatal days (P) 3, 8, 13, 24, and 33 and prepared the cortical brain tissue for immunohistochemistry, in situ hybridization (ISH), Western blot analyses and vessel density counting. When compared to age-matched controls, hypoxic-reared animals displayed a significant increase in platelet endothelial cell adhesion molecule 1 (PECAM-1) protein levels, cerebral microvascular lumen diameter and number and density of vessels (number of capillaries per area). In control animals, ISH and immunohistochemistry revealed that localization of VEGF is restricted almost exclusively to cortical neurons at early stages of development. As the vascular bed begins to stabilize, predominant VEGF expression switches to maturing glial cells which invest vessels while neuronal expression is reduced to a basal level. In hypoxic animals, early localization of VEGF is also restricted to cortical neurons, however, during later developmental stages, glial cells express elevated levels of VEGF protein and high neuronal expression also persists. Thus chronic sublethal hypoxia disrupts the temporal-spatial expression of VEGF, which correlates with continuing hypoxia-driven angiogenesis.

Diffuse nodular eyelid lipogranuloma following sutureless transconjunctival blepharoplasty dressed with topical ointment.

PURPOSE: Transconjunctival blepharoplasty is becoming the approach of choice for many cosmetic surgeons. The authors describe a case of a diffuse, multinodular eyelid lipogranuloma following transconjunctival blepharoplasty, after which the unsutured wound was dressed with a topical ointment. METHODS: Report of clinical course and histopathologic findings. RESULTS: A patient developed multiple firm, nontender masses of the left lower eyelid that enlarged despite topical and systemic medical therapy. Prior to referral, the progressive lesions had recurred despite three successive attempts at surgical eradication. Histopathologic examination of excised tissue demonstrated a multifocal lipogranulomatous inflammation consistent with reaction to retained ointment. CONCLUSIONS: Sclerosing lipogranulomas are a known complication of intradermal lipid injection, as well as a late complication of sinus surgery after postoperative nasal packing with ointment-saturated gauze. The application of a topical ointment should be avoided until after transconjunctival lower blepharoplasty unless wound closure is secure or until conjunctival epithelialization is complete.

Correction of lower eyelid retraction by transconjunctival retractor excision and lateral eyelid suspension.

PURPOSE: To investigate the effectiveness of a procedure that addresses both the lower eyelid retractors and the lateral canthus in the treatment of patients with lower eyelid retraction. METHODS: Through a combined lateral canthotomy and full-length transconjunctival incision, the lower eyelid retractors were disinserted across the horizontal length of the eyelid, recessed to the inferior fornix, and excised. A lateral canthopexy elevated the mobilized eyelid, and horizontal length disparity was corrected. RESULTS: Forty lower eyelid operations in 23 patients yielded good results; all patients attained significant improvement in both eyelid position and function. No reoperations were required during a mean follow-up period of 28 months. CONCLUSION: Although not ideal for severe cases requiring posterior lamellar spacers or anterior lamellar (skin) grafts, this union of techniques successfully treats many types of lower eyelid retraction.

Centurion syndrome and its surgical management.

PURPOSE: To describe the clinical features and treatment outcomes in patients with the centurion syndrome. METHODS: Review of medical records. RESULTS: Forty patients, 38 of whom were male, were examined. Epiphora commenced during the second decade of life in all patients. Fluorescein dye pooled near the medial canthus because of anterior displacement of the medial lower eyelid and inferior punctum. Probing of the canaliculi and irrigation of the lacrimal drainage system confirmed anatomic patency in all patients. Disinsertion of the anterior limb of the medial canthal tendon relieved epiphora by restoring normal apposition between the lower eyelid and the globe in all 22 patients who underwent surgery. CONCLUSION: Centurion syndrome is a unique clinical entity that may manifest as epiphora during puberty. Outcomes of surgical correction are favorable.

Association of chronic sublethal hypoxia with ventriculomegaly in the developing rat brain.

Bronchopulmonary dysplasia remains a major cause of neurodevelopmental handicap in preterm infants. Because bronchopulmonary dysplasia may be associated with prolonged hypoxemia without obvious changes in systemic blood pressure, we developed an animal model of chronic sublethal hypoxia to test the hypothesis that this insult results in significant alterations in corticogenesis in the developing brain. Three groups of newborn rats were placed in a chamber with FIO2 9.5% on postnatal day 3 (P3). One group was sacrificed at P13; a second group was sacrificed at P33, and the third group was removed at P33 and reared in normoxia until sacrifice at P63. Control rats were those raised in room air for the corresponding periods of time. Rats were transcardially perfused and the brains were embedded in celloidin and prepared for morphometric analysis using standard stereology methods. Although experimental rat pups in the third group demonstrated 'catch-up' of body weight following return to normoxia, these studies demonstrated both failure of brain growth (p<0.01) and progressive cerebral ventriculomegaly (p<0.01). Decreased subcortical white matter (p<0. 05) and corpus callosum size (p<0.01) were noted at P63 in pups reared under conditions of chronic hypoxia. Decreases in cortical volume (p<0.05) were noted at all three experimental time points for hypoxic-reared pups when compared to control animals. These data suggest that chronic sublethal hypoxia may lead to severe impairments in corticogenesis in an animal model of developing brain.

Sino-orbital giant cell reparative granuloma.

An otherwise healthy, 9-year-old boy had gradual onset of a mass deformity in the region of the left medial canthus with resulting superior and lateral globe displacement. Radiographic evaluation demonstrated an osteolytic, expansile lesion involving the bones of the ethmoid and maxillary sinuses. Combined anterior orbitotomy and nasal endoscopic surgical resection revealed additional involvement of the nasolacrimal sac wall. Histopathology was consistent with a giant cell reparative granuloma. The authors know of 15 case reports of sino-orbital giant cell reparative granulomas with ophthalmic manifestations; only 3 of these appeared in the ophthalmology literature. This case appears unique in that extension of the abnormal tissue into the nasolacrimal sac wall was demonstrated.

An in vitro three-dimensional coculture model of cerebral microvascular angiogenesis and differentiation.

The microvasculature of the developing brain is plastic and responds differently to the many insults associated with preterm birth. We developed three-dimensional in vitro culture models for the study of the responses of the developing cerebral microvasculature. Beagle brain microvascular endothelial cells (BBMEC) were isolated by differential centrifugation from newborn beagle pups on postnatal Day 1 and placed in three-dimensional culture dispersed in a collagen gel. Alternatively, BBMEC were placed in a three-dimensional coculture with neonatal rat forebrain astrocytes. Cultures were analyzed for extracellular matrix components at 1 and 6 d, and total RNA was extracted for Northern analyses. Urokinase plasminogen activator activity was assayed in both mono- and cocultures of the two cell types. Studies of three-dimensional BBMEC/astrocyte cocultures demonstrated progressive tube formation with only low levels of endothelial proliferation. By 6 d in three-dimensional coculture, the BBMEC formed capillarylike tubes with a wrapping of glial processes, and basement membrane protein synthesis was noted. Urokinase plasminogen zymography suggested intercellular signaling by the two cell types. These data suggest that the three-dimensional beagle brain germinal matrix microvascular endothelial cell/neonatal rat astrocyte coculture provides a good model for the investigation of microvascular responses in the developing brain.

Chronic postnatal hypoxia increases the numbers of cortical neurons.

Premature infants have been shown to undergo prolonged periods of sublethal hypoxia. There is considerable evidence to link these hypoxic events with neurodevelopmental disorders. As an animal model for this clinical problem, rats were raised from the third day of life in a chamber where the O2 level was 9.5%. After 30 days of hypoxia the rats were sacrificed and their brains processed for determination of the number of cortical neurons. This work was performed to test the hypothesis that chronic hypoxia would result in increased cortical cell death. The hypoxic rats had lower body and brain weights as well as decreased cortical volumes. However, hypoxic rats had increased neuronal density and significantly more cortical neurons than controls (P < 0.05). The results of this study suggest that chronic sublethal hypoxia may lead to reduction in the amount of programmed cell death in the developing neocortex.

Vascular endothelial growth factor mediates reactive angiogenesis in the postnatal developing brain.

Although chronic sublethal hypoxia has been shown to promote angiogenesis in the developing brain, the pathogenesis of this response is unknown. We hypothesized that this response may be mediated in part by vascular endothelial growth factor (VEGF). We reared newborn rats (P3) in a chamber with FIO2 of 9.5 +/- 1% (exposed, E). At P33, the animals were removed from the chamber and the brains prepared for immunohistochemistry, mRNA extraction, or horseradish peroxidase (HRP) permeability studies. We also isolated beagle brain germinal matrix endothelial cells from PND 1 beagle pups and placed them in three-dimensional (3-D) coculture with PND 1 rat forebrain astrocytes. Cultures were grown for 6 days in 11% O2 and compared to control 3-D cocultures. When compared to age-matched controls, the experimental rats had significantly increased cortical vascular density (vessels/mm2: 518 +/- 18 vs. 400 +/- 15, P = 0.025). HRP studies demonstrated significantly increased permeability in all cortical vessels examined in experimental rats compared to controls. Compared to controls, VEGF mRNA from hypoxic pups was increased 2.4 times, and immunohistochemical studies of VEGF protein confirmed this finding. Similarly, when compared to controls, hypoxic cocultures of brain microvascular endothelial cells and astrocytes demonstrated significant increase in tubelike structures representing in vitro angiogenesis. Additionally, astrocyte VEGF protein levels increased 4.4-fold in hypoxic compared to control astrocyte cultures and VEGF protein levels increased 1.7-fold in hypoxic compared to control cocultures. Finally, addition of VEGF (10 ng/ml culture medium) to BBMEC alone in 3-D culture elicited not only significant proliferation (P = 0.001) but also increased tube formation. These data demonstrate that the developing brain responds to chronic sublethal hypoxia with increases in permeability and angiogenesis and suggest that VEGF mediates this response.

The individual versus the statistical patient: relationships between treatment of malignant eyelid neoplasms and their "Hosts".

The essence of the doctor-patient interaction is that of a caring relationship. Too often, our reliance upon data, statistical analysis, and out-come percentages obscure the need for subjective interpretation of each healing interaction in which we are engaged. Awareness and responsiveness, on personal and professional levels, to the intuitive and sacred aspects of the doctor-patient relationship need to be recognized as being as important as the rational and scientific aspects of treatment and management if we are to be healers in the fullest sense.

Lotus lectin labels subpopulation of olfactory receptor cells.

Experiments were performed to test the hypothesis that subsets of olfactory receptor cells could be recognized based on their lectin binding and that mapping of their projections onto the olfactory bulb would reveal details of anatomic organization of the olfactory nerve projection to the olfactory bulb. The results from one lectin, Lotus, were examined in detail. Olfactory receptor cells in the lateral part of the main epithelium were labeled, as well as scattered cells in the remainder of the epithelium. Glomeruli labeled by Lotus were concentrated primarily in the region of the olfactory bulb that receives its input from the lateral epithelium, although scattered glomeruli could be identified in other regions. Within the terminal field of these axons there was a mosaic pattern, with some glomeruli densely labeled, some lightly labeled and others unlabeled. These findings support the notion that there are biochemically distinct populations of olfactory receptor cells having localized distributions in the epithelium, with axons that coalesce to terminate in specific glomeruli, rather than diffusely over their projection field.

Germinal matrix microvascular maturation correlates inversely with the risk period for neonatal intraventricular hemorrhage.

The risk period for intraventricular hemorrhage (IVH) of the preterm neonate is the first 3-4 postnatal days. For infants of < 34 weeks' gestation, this risk period is independent of gestational age. We hypothesized that this risk period is attributable to the perinatal induction of maturation of the germinal matrix microvasculature and tested this hypothesis by examining changes in the classical ultrastructural features of the blood-brain barrier over the first ten postnatal days in the newborn beagle model for neonatal IVH. Newborn beagle pups (n = 6) were anesthetized and systemically perfused and the brains were removed and prepared for electron microscopic examination. Examination of electron micrographs from the germinal matrix of animals on the first, fourth and tenth postnatal days demonstrated no difference in perimeter lengths and capillary and endothelial cell areas; in contrast, luminal areas significantly decreased across postnatal age (P = 0.04). Significant increases were found in basement membrane area between days 1 and 4 (P = 0.01) and tight junction length (day 1 vs. day 10, P = 0.02). In addition, on day 1, 19% of germinal matrix capillary perimeter was determined not to be covered by supporting cell processes, while by day 10, only 5% was bare. In contrast, the microvessels of the white matter exhibited no changes in these parameters during these three time points. These studies are consistent with the concept that basal lamina deposition and organization precede increases in endothelial cell tight junction formation and coverage by supporting cells.

Indomethacin promotes germinal matrix microvessel maturation in the newborn beagle pup.

BACKGROUND AND PURPOSE: Although indomethacin has been demonstrated to prevent germinal matrix and intraventricular hemorrhage in clinical and animal studies, the mechanism of action of this agent to prevent hemorrhage remains unclear. Previous studies have demonstrated both that the microvessels in the germinal matrix of newborn beagle pups undergo basement membrane maturation during the first 4 postnatal days and that indomethacin may promote laminin deposition in tumor cell culture systems. METHODS: We employed the newborn beagle pup model to test the hypothesis that indomethacin may stimulate laminin deposition in germinal matrix microvessels. Newborn pups were randomized to receive either 0.1 mg/kg/dose i.p. indomethacin or an equal volume of saline diluent. Pups received doses of study medication once a day for 1, 2, or 3 days and were studied on postnatal days 1, 2, 3, or 4. Pups were anesthetized and systemically perfused with buffered formalin; the brains were removed and prepared for immunohistochemical study. RESULTS: Sections stained with Bandeiraea lectin demonstrated that there was no difference in germinal matrix vessel density among the postnatal ages studied; similarly, there were no differences in vessel density between saline- and indomethacin-treated animals at any postnatal age. Quantification of germinal matrix stained intensity by confocal microscopy demonstrated significant increases in indomethacin-treated pups for both laminin staining at postnatal days 2 (p = 0.05) and 3 (p = 0.0009) and type V collagen staining at postnatal day 2 (p = 0.011). Although staining for beta 1 integrins increased across postnatal ages, there were no differences between saline- and indomethacin-treated animals. CONCLUSIONS: These data suggest that indomethacin may stimulate basement membrane deposition in the germinal matrix microvessels of newborn beagle pups to prevent germinal matrix and/or intraventricular hemorrhage.

Temporoparietal fascial flap for orbital and eyelid reconstruction.

The temporoparietal fascial flap is a recognized technique for the transfer of vascularized tissue in the craniofacial region. The flap has a predictable axial vessel, provides thin vascularized tissue, and can be harvested with minimal donor-site morbidity. The temporoparietal fascial flap is well suited for orbital or eyelid reconstruction because of its proximity to the orbit. The flap is useful for reconstruction of anatomic barriers between the orbit, intracranial cavity, and paranasal sinus spaces. We present four patients in whom the temporoparietal fascial flap was used for orbital reconstruction following extirpative surgery for orbital neoplasm and two patients in whom the flap was used for lower eyelid and malar reconstruction.