Evaluation of cell morphology and adhesion capacity of human gingival fibroblasts on titanium discs with different roughened surfaces: an in vitro scanning electron microscope analysis and cell culture study.

BACKGROUND: Implantoplasty is an option in peri-implantitis treatment. What is known about the effects of implantoplasty on peri-implant soft tissue adhesion and cell behaviours is limited. This study aimed to evaluate the morphological features and adhesion capacity of human gingival fibroblast (HGF) cells onto sand-blasted, large-grit, acid-etched (SLA®) titanium (Ti) discs surfaces roughened with different implantoplasty protocols. MATERIALS AND METHODS: The study included a total of 48 Ti discs divided into four groups (n = 12 per group): Group I: machined, smooth surface discs; Group II: SLA® surface discs; Group III: SLA® surface discs roughened with diamond bur sequence (40 and 15-µm grit); Group IV: SLA® surface discs roughened with diamond bur sequence (125 and 40-µm grit). Following polishing procedure, the surface roughness value of discs was assessed by a profilometer and scanning electron microscope. HGFs were cultured on Ti discs and cell adhesion was examined after the 24th, 48th, and 72nd hours. Statistical significance was set at the p ≤ 0.05 level. RESULTS: Scanning electron microscope analyses of the discs revealed that fibroblasts exhibited well-dispersion and a firm attachment in all groups. The cells in group I and II had thin and long radial extensions from the areas where the nucleus was located to the periphery; however, attached cells in group III and IV showed more spindle-shaped morphology. The surface roughness parameters of the test groups were lower than those of the SLA®. The SLA® group showed the highest HGF adhesion (group II) (p ≤ 0.05). HGF adhesion in group IV was greater compared to group III, but less than group I. CONCLUSIONS: This study showed that the characteristics of the burs applied in the implantoplasty protocol are determinant for the surface roughness and fibroblast adhesion occurs on surfaces with decreased roughness following implantoplasty. Consequently, it should be kept in mind that the surface properties of the implant may affect the adherent cell morphology and adhesion.

Placental expression of insulin-like growth factor-I, fibroblast growth factor-basic and neural cell adhesion molecule in pregnancies with small for gestational age fetuses.

OBJECTIVE: To investigate placental expression of insulin-like growth factor-I (IGF-I), fibroblast growth factor-basic (FGF-b) and neural cell adhesion molecule (N-CAM) regarding the pathogenesis of pregnancies with small for gestational age (SGA) fetuses. STUDY DESIGN: An immunohistochemical analysis using anti-IGF-I, anti-FGF-b and anti-N-CAM antibodies was carried out on 4% paraformaldehyde-fixed placental tissues of third trimester pregnancies complicated with SGA fetuses (n=12) and subjects exhibiting appropriately grown fetuses (n=10). Immunostaining patterns of chorionic villi and amniochorionic membranes were assessed. RESULT: IGF-I, FGF-b and N-CAM immunostainings in chorionic villi demonstrated significantly increased immunoreactivities in cytotrophoblasts of SGA cases, whereas increased IGF-I immunostaining in syncitiotrophoblasts and increased N-CAM immunostaining in capillary endothelium were noted in the same group. IGF-I, FGF-b and N-CAM immunostainings in amniochorionic membranes revealed significantly decreased IGF-I immunoreactivities in extravillous trophoblasts and increased IGF-I immunoreactivities in decidual cells of SGA cases, while significantly decreased N-CAM immunoreactivities in both decidual cells and extravillous trophoblasts were noted. FGF-b immunostaining revealed no significant differences in both extravillous trophoblasts and decidual cells of SGA cases. CONCLUSION: Increased placental expression of IGF-I, FGF-b and N-CAM may act in an autocrine and/or paracrine manner to restore the impaired trophoblastic proliferation, migration and metabolism at all gestational stages by means of a positive feedback mechanism.

The cardioprotective effects of thoracal epidural anestesia are induced by the expression of vascular endothelial growth factor and inducible nitric oxide synthase in cardiopulmonary bypass surgery.

AIM: The cardioprotective effects of thoracal epidural anesthesia (TEA) are induced by the expression of vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (i-NOS) in cardiopulmonary bypass (CPB) surgery. When general anaesthesia (GA) is combined with TEA during coronary artery bypass graft, we investigated whether TEA together with GA play a role on VEGF and i-NOS expression in human heart tissue in cardiac ischemia. METHODS: Right atrial biopsy samples were taken before CPB, before aortic cross clamp (ACC) and at 15 min after ACC release (after ischemia and reperfusion). Human heart tissues were obtained from the TEA+GA and GA groups. Immunocytochemistry was performed using antibodies for VEGF and i-NOS. RESULTS: Both VEGF and i-NOS immunoreactivity was observed in cardiomyocytes and arteriol walls. Although VEGF and i-NOS immunoreactivity was apparent in both groups,, immunostaining intensity was greater in the TEA+GA group than the GA group. Between groups, at 4 h and at 24 h after the end of CPB, the cardiac index (CI) was significantly higher in the TEA+GA group than GA group (3.4+/-0.8 L/min/m(2) vs 2.5+/-0.8 L/min/m(2); P<0.001), (3.8+/-1.1 L/min/m(2) vs 3.1+/-1.1 L/min/m(2); P<0.008) respectively. Within groups, at 4 and 24 h after the end of CPB, the CI was significantly higher in the TEA+GA group than baseline values, (3.4+/-0.8 L/min/m(2) vs 2.4+/-0.7 L/min/m(2); P<0.001), (3.8 +/-1.1 L/min/m(2) vs 2.4+/-0.7 L/min/m(2); P<0.001) respectively, but no difference was found in the GA group (2.6+/-0.8 L/min/m(2) vs 2.5+/-0.8 L/min/m(2); P>0.05), (2.6+/-0.8 L/min/m(2) vs 3.1+/-1.1 L/min/m(2); P>0.05) respectively. After ACC release, 11/40 (27.5%) patients in the TEA+GA group showed ventricular fibrillation (VF), atrial fibrillation or heart block versus 25/40 (62.5%) of those in the GA group. VF after ACC release in the TEA+GA group (9/20 patients, 22.5%) was significantly lower than in the GA group (21/40 patients, 52.5%); (P<0.006). Sinus rhythm after ACC release in the TEA+GA group (29/40 patients, 72.5%) was significantly higher than in the GA group (15/40 patients, 37.5%); (P<0.002). CONCLUSIONS: The results of the present study indicate that TEA plus GA in coronary surgery preserve cardiac function via increased expression of VEGF and i-NOS, improved hemodynamic function and reduced arrhythmias after ACC release.

General anesthesia with thoracic epidural anesthesia in the cardiopulmonary bypass surgery reduces apoptosis by upregulating antiapoptotic protein Bcl-2.

AIM: The aim of the paper was to investigate whether thoracic epidural anesthesia (TEA) together with general anaesthesia (GA) play a role on apoptosis in humans before cardiopulmonary bypass (CPB), before aortic cross clamp (ACC) and at 15 min after ACC release (after ischemia and reperfusion). METHODS: Eighty patients scheduled for elective CABG were randomized to receive either GA group (n: 40) or TEA+GA group (n: 40). The right atrial biopsy samples were taken before CPB, before ACC and at 15 min after ACC release from all patients. Human heart tissues were obtained from patients of TEA+GA group and GA group. The number of Bcl-2 positive cardiomyocytes was counted in multiple tissue sections of biopsies of 80 patients using light microscopy (magnification x 40) with an ocular micrometer system (Olympus). RESULTS: In the TEA+GA group, the Bcl-2 positive cardiomyocytes were distinctly statistically increased compared to the GA group (P<0.001). In addition, the intensity of the immunostaining was also increased in the TEA+GA compared with the GA group. The number of immunoreactive cardiomyocytes is as follows: before CPB, TEA+GA group 396+/-61, GA group 92+/-41, before ACC, TEA+GA group 333+/-47, GA group 94+/-18, at 15 min after ACC release, TEA+GA group 346+/-68.8, GA group 85+/-9.5. There were statistically significant differences between groups, (P<0.001). Between groups, at 4 h and at 24 h after the end of CPB, in the TEA+GA group, the CI was significantly higher than GA group respectively; (3.4+/-0.8 L/min/m(2) vs 2.5+/-0.8 L/min/m(2); P<0.001), (3.8+/-1.1 L/min/m(2) vs 3.1+/-1.1 L/min/m(2); P<0.008). Within groups, at 4 and 24 h after the end of CPB, in the TEA+GA group, the CI was significantly higher than baseline values, respectively, (3.4+/-0.8 L/min/m(2) vs 2.4+/-0.7 L/min/m(2); P<0.001), (3.8+/-1.1 L/min/m(2) vs 2.4+/-0.7 L/min/m(2); P<0.001). Whereas no difference was found in the GA group respectively, (2.6+/-0.8 L/min/m(2) vs. 2.5+/-0.8 L/min/m(2); P>0.05), (2.6+/-0.8 L/min/m(2) vs. 3.1+/-1.1 L/min/m(2); P>0.05). The number of patients showing ventricular fibrillation (VF), atrial fibrillation or heart block after release of the ACC was 11 of 40 (27.5%) in the TEA+GA group versus 25 of 40 (62.5%) in the GA group. The number of patients showing VF after release of ACC was 9 out of 20 patients (22.5%) in the TEA+GA group which was significantly lower than in the GA group (21 of 40 patients 52.5%); (P<0.006). Sinus rhythm after release of ACC, in the TEA+GA group was observed in 29 of 40 patients (72.5%) and was significantly higher than in the GA group (15 of 40 patients 37.5%); (P<0.002). CONCLUSION: The result of the present study indicate that TEA plus GA in coronary surgery had preserved cardiac function during intraoperative and postoperative period by means of reduced apoptosis, improved hemodynamic function and reduced arrhythmias after release of the ACC.

The effects of ovulation induction on ovarian epithelium dysplasia scores and Ki67 expression: an experimental study on rats.

We aimed to evaluate the effects of ovulation induction on Ki67 expression and dysplasia scores of female rat ovaries. Twenty female Wistar rats were randomized either to receive 150 IU/kg human menopausal gonadotropin on estrous day 2 and 75 IU/kg human chorionic gonadotropin on the day of preestrous (induction group, n= 10) or saline as placebo on the corresponding days (control group, n= 10). After five estrous cycles bilateral oophorectomy was performed to compare the Ki67 expression and dysplasia score of the ovarian epithelium. The mean number of the cells that stained positive for Ki67 was 159.6 +/- 101.92 in the follicles, 283.4 +/- 42 in the corpus luteum, and 151 +/- 75.1 in the stroma of the study group compared to 41.8 +/- 35.6 (P= 0.03), 43.2 +/- 28.3 (P= 0.007), and 55.6 +/- 18.6 (P= 0.01), respectively, in the control group. The mean number and rate of cells that stained positive for Ki67 in the epithelium was significantly higher in the ovulation induction group (758 +/- 71 and 63 +/- 1.6%, respectively) compared to the control group (386 +/- 23, P < 0.001; and 60 +/- 1.1%, P < 0.001; respectively). The mean dysplasia score was significantly higher (9.6 +/- 1.3) in the study group compared to the control group (5.08 +/- 0.9, P < 0.001). Ovulation induction in rats resulted in increased Ki67 expression and dysplastic features in the ovarian epithelial cells.

Effect of insulin-like growth factor-1 on apoptosis of rat testicular germ cells induced by testicular torsion.

OBJECTIVE: To investigate the possible protective role of insulin-like growth factor-1 (IGF-1, reported to have a protective effect in experimental models of hypoxic ischaemia), and the involvement of apoptotic cell death in a model of torsion/detorsion of the rat testis. MATERIALS AND METHODS: Adult male Wistar rats were divided into five groups of five rats each. Group 1 underwent a sham operation as a control; in group 2 the testis was twisted and in group 3 then untwisted; in group 4 IGF-1 was injected subcutaneously just before bilateral torsion, and then the right testis removed after 4 h and the left after 24 h; in group 5, IGF-1 was injected immediately after bilateral detorsion and then the testes removed as in group 4. Both testicles were examined histologically, with apoptosis detected using the in situ DNA fragmentation (TUNEL) system, with combined enzymology and immunohistochemistry techniques. RESULTS: In groups 2 (torsion) and 3 (detorsion), light microscopy of the testis showed some degenerative changes in the germ cells. Compared to group 1, apoptosis was more significant in group 3 than in the other groups. Group 4 (torsion/IGF-1) had a similar number of apoptotic germ cells as in group 2 (torsion) after 24 h, but fewer than the same group after 4 h. In group 5 (detorsion/IGF-1), apoptosis was reduced by IGF-1 significantly more than in group 3 (P < 0.05). Apoptosis was significantly less in spermatids in group 5 than in group 3 (P < 0.05). CONCLUSIONS: IGF-1 seems to lower the levels of germ cell apoptosis, which may be important for protecting the testes from torsion/detorsion injury. Further clinical studies are needed to evaluate this protective effect in testicular torsion/detorsion.

Localization of neural cell adhesion molecule (N-CAM) immunoreactivity in adult rat tissues.

Neural cell adhesion molecule (N-CAM) mediates homophilic adhesion between cells and heterophilic adhesion between cells and extracellular matrix in a Ca2+-independent manner. N-CAM is widely expressed during development and plays a crucial role in cell division, migration, and differentiation, but its expression is restricted in adults. The distribution of N-CAM immunoreactivity in adult rat tissues was investigated in the present study. N-CAM immunoreactivity was present in the nervous system in the molecular layer of the cerebellum, ependymal cells surrounding the central canal, axons of the white matter, and in Lamina X of the gray matter of the spinal cord. N-CAM immunoreactivity also was found in autonomic nerves. In the digestive system, N-CAM immunoreactivity was found in the stratified squamous epithelium and nerve plexus of the esophagus, glandular cells of the stomach and pylorus, lamina propria, and epithelium of the villi of the duodenum, jejunum, and ileum. N-CAM immunoreactivity was demonstrated in the secretory cells of the adenohypophysis, islets of Langerhans, and acinar cells of the exocrine pancreas. Alveolar cells of the lung were also N-CAM immunoreactive. In the urinary system, N-CAM immunoreactivity was seen in the proximal convoluted tubules of the kidney. In the male reproductive system, N-CAM immunoreactivity was demonstrated in the nerve plexus around the urethral epithelium and in the nerve fibers around the smooth muscle cells of the corpus cavernosum penis. In the visual system, N-CAM immunoreactivity was seen in the epithelial cells of the corpus ciliaris. Cornea and lens epithelium also showed positive immunoreactivity. Our results suggest that cells in many tissues and organs of the adult rat synthesize N-CAM.

Expression of insulin-like growth factor in the placenta of intrauterine growth-retarded human fetuses.

Many cases of intrauterine growth retardation (IUGR) are the result of placental and fetal tissue insufficiency. Insulin-like growth factor-I (IGF-I) is known to play a role in placental and fetal growth. An immunocytochemical study was performed to localize IGF-I peptides in human placenta and umbilical cords of normal (n = 3) and IUGR (n = 3) fetuses. The peripartum fetal conditions were evaluated as well. Immunoreactive IGF-I was detected in the cytotrophoblast, syncytiotrophoblast, amnion, endothelial cells of fetal capillaries and in the decidua in both normal and IUGR placental tissue. A more robust immunostaining and increased numbers of positively stained cells were found in the decidua of IUGR placenta (p < 0.001). Intense immunostaining was also found in endothelial cells, smooth muscle cells and fibroblasts of the umbilical vein. IGF-I immunoreactivity was also present in stroma (Hofbauer cells and/or fibroblasts) of IUGR villi. Our results indicate that expression of IGF-I is high in specific sites in placenta and umbilical cords, which indicates a paracrine and/or endocrine function. The increased expression of IGF-I in placenta of IUGR fetuses indicates its involvement in restoring normal growth by means of a positive feed-back mechanism.

Antiproliferative effects and insulin-like growth factor-I expression in Balb-C 3T3 fibroblasts after treatment with somatostatin and gonadotropin-releasing hormone analogs.

The present study was aimed to compare antiproliferative effects of somatostatin (SS) and gonadotropin-releasing hormone analogs (GnRHa) on a fibroblast cell line. Proliferation index, cell count, viability of the cells and insulin-like growth factor-I (IGF-I) immunoreactivity were determined after treatment with either SS (100 microM/ml), GnRHa (35 nM/ml) or SS and GnRHa of Balb-C 3T3 mouse fibroblasts. It was found that the proliferation index, cell count, viability and IGF-I immunoreactivity were not affected by GnRHa treatment as compared with no treatment (p > 0.05). Application of SS to the fibroblasts resulted in a significant reduction in proliferation index, cell count, and IGF-I immunoreactivity as compared with GnRHa treatment and no treatment, but it had no effect on cell viability. The labelling index in SS-treated cells was significantly reduced as compared with combined treatment with SS and GnRHa. In conclusion, a direct effect of GnRHa on fibroblast cells in culture could not be demonstrated. SS had direct inhibitory effects on cell proliferation possibly via inhibition of IGF-I effects without affecting cell viability.

Unusual origin of the right renal artery: a case report.

An anomalous origin of the right renal artery was observed in a 50-year-old male cadaver. The importance of this variation of the right renal artery and superior mesenteric artery arising from a common trunk is emphasised.

Protective effects of vitamin E and selenium on the renal morphology in rats fed high-cholesterol diets.

The histopathological effects of cholesterol and the protective effects of vitamin E and selenium (Se) on renal histology were examined in Sprague-Dawley rats. Light-microscopic evaluation of the renal cortex revealed: glomerular fibrosis, cellular and mesangial proliferation, capillary obliteration and cholesterol crystals in the tubular lumina of the cholesterol-fed group. These results suggest that oxidated LDL (O-LDL) is a cytotoxic factor which stimulates mesangial cell and matrix proliferation. Ultrastructurally, small and large lipid vacuolization in intracapillary lumina, adhesion of epithelial foot processes, mesangial foam cells and polymorphonuclear leukocytes were seen in the cholesterol-fed group. In the groups fed cholesterol + vitamin E, cholesterol + Se and cholesterol + vitamin E + Se, morphological improvements were observed. It appeared that an excess in O-LDL, reactive oxygen species and growth factors might play an important role in the pathogenesis of glomerulosclerosis. In addition, it was concluded that antioxidant therapy may prevent LDL oxidation and generation of free radicals.

Multiple variations of the azygos venous system.

In a dissection performed in our department, we observed multiple variations of the azygos venous system. The hemiazygos vein was absent. The posterior 8th, 9th, and 10th intercostal veins united and their common trunk crossed the vertebral column obliquely lying anterior to the aorta and posterior to the esophagus and opening into the azygos vein at the level of T7-T8 vertebrae. The 7th left posterior intercostal vein also crossed the column anteriorly and joined the common trunk. The present report identifies the variable positions and courses of the veins related to the azygos system. It is important to keep in mind that different courses of the azygos system do exist, so that extra caution is required during surgery of the mediastinum and also in appropriately interpreting the radiographs.

The anatomy of lamina pretrachealis fasciae cervicalis.

The definitions concerning the fascia pretrachealis is either contradictory or insufficient in anatomy textbooks. The fascia pretracheatis is clinically important in the procedure of tracheostomy, mediastinascopy and also in tracheal and bronchial trauma. The anatomy of the fascia pretrachealis (extension, relation and the attachments) was reexamined using cadaveric preparations and the clinical value of the fascia is reinforced. The fascia pretrachealis is attached to the upper brim and to the oblique line of the thyroid cartilage and continued its course on the anterior surface of the trachea and fused with the advantitia of arch of the aorta, posterior aspect of pulmonary artery and the pericardium. Laterally it is attached to the cartilagenous part of the trachea. Also contraversial literature concerning description of the fascia pretrachealis has been evaluated.

A morphological study on the V2 segment of the vertebral artery.

Morphometric investigations on the V2 segment of the vertebral artery, showed that, it did not have a constant calibre during its course within the foramina transversaria. The vertebral artery, entering the foramina transversaria reduced its calibre and further continued to reduce until C3 level, above C3 it began to reincrease its calibre and at C1 level reached its largest calibre. Measurements on the muscular thickness, showed an increase as ascending through the foramina transversaria. The widening and narrowing of the vertebral artery within the foramina transversaria was attributed as tortious artery or congenital anomaly. This study showed that it was the normal anatomy of the artery within the foramina transversaria.

The cellular localization of vasoactive intestinal peptide (VIP) in the mouse median eminence by immuno-electron microscopy.

The aim of this study was to examine, by use of pre-embedding immunocytochemistry, the ultrastructural localization of vasoactive intestinal peptide (VIP) immunoreactivity in the mouse median eminence. VIP immunoreactivity was observed in axonal profiles. The VIP-immunoreactive axonal profiles were in close proximity to non-immunoreactive axonal profiles that contained dense granular vesicles and clear vesicles and also to processes of tanycytes. VIP-immunoreactive terminals were observed in the proximity of the perivascular space and in the neuropil. Our results suggest that VIP-immunoreactive axon terminals may possibly interact with other non-immunoreactive axon terminals containing peptide and/or other transmitters at the level of the median eminence or may be released to the portal vasculature thereby to effect anterior pituitary cells.

Ultrastructural changes in rat Paneth and goblet cells after the administration of interferon-alpha.

Electron microscopic examination was performed to show the effects of interferon-alpha (IFN-alpha) on Paneth and goblet cells in rat intestines. After the administration of IFN-alpha (70,000 IU/kg), many cells of both types were depleted of secretory granules and their apical membranes had the deep cavitation that accompanies recent compound exocytotic activity. These results may indicate the involvement of these cells in the inflammatory reactions via IFN-alpha and this model provides a powerful tool to study differential effects of IFN-alpha on Paneth and goblet cells.

Response of the rat mucosal and connective tissue type mast cells to interferon-alpha.

The distribution, density and histochemical subtype of mast cells (mucosal and connective tissue) were studied in the ileum, trachea and skin of rats treated with IFN alpha (70.000 IU/kg) treated rats. Light and electron microscopic procedures were utilized. The total number of mucosal mast cells in the sections of ileum and trachea were markedly increased in the IFN-alpha treated group (ileum: 31.9 +/- 2.2 cells/villuscrypt unit; trachea: 10,355 +/- 264 cells/mm3). However, the number of connective tissue mast cells did not show any significant change in the skin between IFN-alpha treated (1,472 +/- 125 cells/mm3) and saline-treated (1,757 +/- 264 cells/mm3) groups. We conclude that mast cell proliferation does exist in the rat ileum and trachea but no in the skin response to IFN-alpha. We suggest that this model provides a powerful tool to study differential effects of IFN-alpha on mast cell subtypes and to identify their role in the immunoregulatory and inflammatory reactions.

A rare case of the posterior inferior cerebellar artery.

The posterior inferior cerebellar artery (PICA) is normally a branch of the vertebral artery. In this rare case, the unilateral left vertebral artery continued its course as the left PICA, and an extremely small caliber left vertebral artery joined the righ vertebral artery to form the basilar artery. This rare feature of the PICA is demonstrated and its relation to neighboring structures is discussed. Also, the literature concerning the PICA is reviewed.

Growth hormone-releasing hormone peptide and mRNA are overexpressed in GH-deficient Ames dwarf mice.

Hypothalamic expression of growth hormone-releasing hormone (GHRH) was quantified morphologically in dwarf mice which exhibit spontaneous genetic GH absence. Mouse GHRH mRNA was assessed by in situ hybridization; densitometric evaluation of total mRNA in dwarfs showed levels 2.3-fold higher than in phenotypically normal siblings (p < 0.01); assessment of mRNA per neuron by autoradiographic grain counting indicated a 2.5-fold increase per cell in dwarfs (p < 0.005). GHRH peptide was evaluated immunocytochemically using a new mouse-specific antiserum; numbers of neurons containing detectable levels were 3-fold higher in dwarfs (p < 0.005). The increase in GHRH mRNA corroborates that reported previously in the GH-deficient little mouse, and after hypophysectomy in rats; GHRH peptide increase contrasts with previous reports of the effect of acute GH removal by hypophysectomy, in which GHRH levels fell. The results suggest that chronic GH deficiency is accompanied by increased translation as well as transcription of GHRH.

Median eminence-afferent vasoactive intestinal peptide (VIP) neurons in the hypothalamus: localization by simultaneous tract tracing and immunocytochemistry.

Retrograde tract tracing and immunocytochemistry were used to investigate the CNS source of the VIP that is present in high concentrations in the hypophysial portal blood and has been shown to have a stimulatory effect on pituitary prolactin secretion. Fluoro-gold (FG), which enters the CNS through areas devoid of the blood-brain barrier, such as median eminence, was injected peripherally. Brain sections from FG-treated animals were immunostained for VIP. A small population of VIP-containing cell bodies in the parvocellular and periventricular parts of the paraventricular nucleus (PVN) was also labeled with FG. Vasoactive intestinal peptide-immunoreactive perikarya not labeled with FG were also observed in the PVN, as well as FG-labeled cells that did not contain VIP. The results suggest that some VIP-producing neurons in the PVN project to the median eminence and are, therefore, functionally related to pituitary regulation; the function of other VIP neurons in the PVN is unknown.