Muristerone A-induced nerve growth factor release from genetically engineered human dermal fibroblasts for peripheral nerve tissue engineering.

In this study, human dermal fibroblasts (hDFBs) were genetically modified to release human nerve growth factor (NGF) using an ecdysone-inducible system. NGF cDNA was inserted into the pIND vector and then hDFBs were cotransfected with pIND-NGF and pVgRXR. Muristerone A, an analog of ecdysone, was used as the inducing agent. NGF release from transfected hDFBs was assessed in vitro and in vivo. Transfected hDFBs in the presence of Muristerone A possessed a maximal in vitro release of 8.5 +/- 0.4 pg of NGF/mL per 10(3) cells, demonstrating significantly higher NGF levels compared to control hDFBs. The in vitro release rate curve for transfected hDFBs in the presence of Muristerone A exhibited a maximum of 5.1 +/- 0.2 ng NGF/10(6) cells/day. A PC-12 bioassay demonstrated that the in vitro NGF released is bioactive. When transfected hDFBs in the presence of Muristerone A were placed in vivo in nude rats, NGF levels reach 2074 +/- 257 pg/mL and 1620 +/- 132 pg/mL at 24 and 48 h, respectively. These levels were significantly higher than negative control and wound fluid levels. Results support further in vivo investigation of this molecular "on" switch for peripheral nerve regeneration.

Incidence of hematoma associated with ketorolac after TRAM flap breast reconstruction.

Ketorolac is frequently used as an adjunct for postoperative pain relief, especially by anesthesiologists during the immediate postoperative period. It can be used alone as an analgesic but is more often used to potentiate the actions of narcotics such as morphine or meperidine in an attempt to reduce the total dose and side effects of those drugs. The manufacturer of ketorolac cautions against its use in patients who have a high risk of postoperative bleeding, for fear of increasing the risk of hematoma, but the risk in transverse rectus abdominis musculocutaneous (TRAM) flap patients has never been reported. In a study of 215 patients who had undergone TRAM flap breast reconstruction, it was determined that patients who received intravenous ketorolac (n = 65) as an adjunct to their treatment with morphine administered by use of a patient-controlled analgesia device required less morphine (mean cumulative dose, 1.39 mg/kg) than did patients who did not receive ketorolac (n = 150; mean cumulative dose, 1.75 mg/kg; p = 0.02). There was no increase in the incidence of hematoma in patients who were treated with ketorolac. The data presented in this study suggest that the use of intravenous ketorolac does reduce the need for narcotics administration in patients undergoing TRAM flap breast reconstruction, without significantly increasing the risk of hematoma.

Postoperative morphine requirements of free TRAM and DIEP flaps.

In a review of the charts of 158 patients who had undergone breast reconstruction with free transverse rectus abdominis musculocutaneous (TRAM) or deep inferior epigastric perforator (DIEP) flaps and who were treated for postoperative pain with morphine administered by a patient-controlled analgesia pump, the total dose of morphine administered during hospitalization for the flap transfer was measured. Patients whose treatment was supplemented by other intravenous narcotics were excluded from the study. The mean amount of morphine per kilogram required by patients who had reconstruction with DIEP flaps (0.74 mg/kg, n = 26) was found to be significantly less than the amount required by patients who had reconstruction with TRAM flaps (1.65 mg/kg; n = 132; p < 0.001). DIEP flap patients also remained in the hospital less time (mean, 4.73 days) than did free TRAM flap patients (mean, 5.21 days; p = 0.026), but the difference was less than one full hospital day. It was concluded that the use of the DIEP flap does reduce the patient requirement for postoperative pain medication and therefore presumably reduces postoperative pain. It may also slightly shorten hospital stay.

Phospholipase Cgamma1 in bovine rod outer segments: immunolocalization and light-dependent binding to membranes.

We have investigated the isozymes of a phosphoinositide-specific phospholipase C (PLC) in bovine retina using several monoclonal antisera to PLCbeta1, gamma1, and delta1. Immunoblot analysis showed that all three isozymes were present in the retina. Immunocytochemical localization in frozen bovine retina sections showed that PLCgamma1 was present in the photoreceptor cell layer, outer plexiform cell layer, inner plexiform cell layer, and ganglion cell layer. Immunoreaction within the photoreceptor cell layer was dependent on dark/light adaptation state of retinas. Immunoblot analysis of rod outer segments (ROS) with monoclonal or polyclonal antibodies to PLCgamma1 showed the presence of an immunoreactive band of 140 kDa. ROS prepared from retinas light-adapted in vitro had more PLCgamma1 on immunoblots than ROS from dark-adapted retinas. PLC enzyme activity in ROS from light-adapted retinas was 69 and 46% higher than ROS from dark-adapted retinas, when assayed in the presence and absence of ATP, respectively. This increase in enzyme activity was observed at [Ca2+]free between 0.32 and 100 microM. These results demonstrate the presence of PLCgamma1 in bovine ROS and show that ROS prepared from light-adapted retinas are enriched in this isozyme, suggesting that light may promote the binding of this isozyme to bleached ROS membranes.

Effect of dietary fat and environmental lighting on the phospholipid molecular species of rat photoreceptor membranes.

We have previously shown that retinas of albino rats adapt to bright cyclic light (500-800 lx) by lowering the levels of docosahexaenoic acid (22:6n-3) in their rod outer segment (ROS) phospholipids. In the present study, we addressed the role of dietary fat in this process. Pregnant rats were kept in 1 lx or 250 lx cyclic illuminance (12L:12D) and fed diets containing 10% (by weight) of either hydrogenated coconut oil (COC, no n-3 or n-6 fatty acids), linseed oil (LIN, n-3 and n-6 fatty acids), or safflower oil (SAF, only n-6 fatty acids), starting 4 days before delivery. Pups were weaned at 3 weeks of age and continued on the same diet and light regime. At 12 weeks of age, 3 or 4 animals in each diet-light group were killed and the remaining animals were stressed continuously with 2000 lx light for 24 hr and then kept in 1 lx cyclic light for 10 days. Fatty acids and phospholipid molecular species (PLMS) of ROS membranes were determined. For prestressed groups, those animals fed the LIN diet had high levels of 22:6n-3 and PLMS containing 22:6n-3, with little 22:5n-6. Compared to the LIN group, the COC and SAF groups had lower levels of 22:6n-3- and 22:6n-3)-containing PLMS and higher levels of 22:5n-6 and molecular species containing 22:5n-6, such as 22:5n-6/22:6n-3, 16:0/22:5n-6 and 18:0/22:5n-6. Within each dietary group, animals raised in 250 lx cyclic illuminance had lower levels of 22:6n-3 and 22:5n-6 compared to those raised at 1 lx. This light effect was greater for 22:6n-3 in the LIN group than for 22:5n-6 in the SAF group. After the acute light stress, those animals raised in 1 lx showed dramatic reduction in PLMS containing 22:6n-3 and 22:5n-6, especially polyenoic species such as 22:6n-3/22:6n-3 in the LIN group and 22:5n-6/22:6n-3 in the COC and SAF groups. In contrast, animals raised in 250 lx showed much smaller changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of dietary fat on the response of the rat retina to chronic and acute light stress.

We designed an experiment to study the role of light history and polyunsaturated fatty acids (PUFA) on the susceptibility of the albino rat retina to light damage. Albino rats were born to dams that had been kept in either 1 lx or 250 lx cyclic light for 0-4 days prior to delivery and fed one of three diets containing either 10% (by weight) hydrogenated coconut oil (COC, no n-3 nor n-6 fatty acids), safflower oil (SAF, high n-6, < 0.1% n-3 fatty acids), or linseed oil (LIN, high n-3, low n-6 fatty acids). After weaning, the rats were maintained in the same light environment and fed the same diets for 9 weeks, at which time some were killed and their retinas processed for morphometric analysis. Animals raised in bright cyclic light had shorter ROS lengths and thinner outer nuclear layers (ONL) than rats raised in dim cyclic light. The LIN animals had a thinner ONL than animals of the SAF or COC groups. Rats from each diet and light rearing groups were exposed to constant illumination of 2000 lx for 24 hr, after which they and non-exposed controls were placed in 1 lx cyclic light for 10 days and analysed for changes in ONL thickness. In the 250 lx (bright; B) groups, there was no effect of acute light stress on ONL thickness, although both control and stressed LIN animals had a thinner ONL than the corresponding COC and SAF groups. However, in the rats raised in 1 lx cyclic light, acute stress resulted in significant retinal damage (i.e. decrease in ONL thickness) in the three diet groups combined. The superior region was damaged the most and the severity was dependent on diet, as evidenced by the LIN group having a greater reduction in ONL thickness than the SAF group after light stress. From these results we conclude that rats on diets high in n-3 fatty acids are more susceptible to photoreceptor cell loss than animals fed n-6 or no polyunsaturated fatty acids when raised in dim cyclic light. These results show that both diet and light history play a role in the susceptibility of the retina to acute and chronic effects of light and suggest a role for lipid peroxidation in retinal light damage.

Immunolocalization of PKC zeta in rat photoreceptor inner segments.

We have utilized several peptide specific antisera directed against the C-terminals (Wetsel et al, 1992) of several protein kinase C (PKC) isozymes (alpha, beta 1, beta 11, gamma, delta, epsilon, zeta) to delineate the cellular localization of these PKC isozymes in rat retina. Antisera against PKC beta 1, beta 11, gamma, delta and epsilon were non-reactive in frozen rat retina sections, whereas, anti PKC alpha was strongly reactive with the outer plexiform, inner plexiform and nerve fiber cell layers. The most specific localization of immunoreactivity was observed with PKC zeta, which reacted strongly and exclusively with photoreceptor inner segments, but not outer segments. Immunoblot analysis of whole rat retina homogenate showed that anti-PKC alpha recognized an antigen of approximately 80kD and anti-PKC zeta recognized a approximately 72kD protein. Immunolocalization of PKC zeta to photoreceptor inner segments and possible functional significance are discussed.

Inositol-1,4,5-trisphosphate receptors in the vertebrate retina.

Evidence has shown an activation of phosphatidylinositol 4,5-bisphosphate (PIP2) specific phospholipase C (PtdIns-PLC) by light in the vertebrate retina and rod outer segments (ROS), suggesting important roles for its two metabolites, 1,2-diacylglycerol (DG) and inositol-1,4,5-trisphosphate [Ins(1,4,5)P3]. DG activates protein kinase C (PKC) and Ins(1,4,5)P3 releases bound intracellular calcium. Since Ca2+ plays an important role in light adaptation, the presence of Ins(1,4,5)P3 receptors in ROS may indicate a regulatory role of Ins(1,4,5)P3 to the free Ca2+ content. In the present study, we investigated the Ins(1,4,5)P3 receptors in whole retinal membranes and several subcellular fractions prepared from bovine retinas. Scatchard analyses of binding data for retinal membrane preparations showed a single, high-affinity binding site with equilibrium dissociation constant (Kd) of 24 +/- 2 nM and maximal binding capacity (Bmax) of 353 +/- 15 fmol/mg protein at pH 7.4. Specific binding was found in both small and large synaptosomal preparations representing inner and outer plexiform layers, respectively. A detectable, but low abundance of Ins(1,4,5)P3-specific binding in ROS was observed at both pH 7.4 and 8.3, but no specific binding of Ins(1,4,5)P3 was found in isolated outer segment discs. The binding of Ins(1,4,5)P3 in ROS was reduced by addition of ATP, suggesting a regulatory role for this nucleotide. Addition of calcium, sodium, and potassium ions also reduced specific binding of Ins(1,4,5)P3. Immunocytochemical studies indicate intense staining in the inner segment and extending to the ROS. Inner and outer plexiform layers were also stained. These findings show that the Ins(1,4,5)P3 receptor is present in photoreceptor cells and inner and outer plexiform layers in the vertebrate retina.

A method for the isolation of retinal pigment epithelial cells from adult rats.

PURPOSE: Isolating retinal pigment epithelium (RPE) cells from adult rat was the goal of this study. METHOD: A modification of the procedure of Mayerson et al was the method employed. After the cornea and lens were removed, the eyecup was treated with hyaluronidase plus collagenase. The neural retina was carefully removed with little RPE attached. The eyecup, free of retina, was treated with the same enzyme cocktail that loosens any attached rod outer segments as well as the attachment of the RPE to Bruch's membrane. RPE cells were isolated in calcium-free medium as sheets of cells. RESULTS: Light and electron microscopy revealed good cell morphology. Purity of the RPE preparation was established by microscopy, polyacrylamide gel electrophoresis, and lipid analysis. The viability of the isolated RPE was about 74% by trypan blue exclusion test. CONCLUSIONS: This technique enables RPE to be isolated from adult rat in quantities sufficient for biochemical analysis.

Oxygen-induced retinopathy in the rat: hemorrhages and dysplasias may lead to retinal detachment.

Rearing neonatal rats in hyperoxia induces the development of retinal hemorrhages and retinal dysplasia. Albino rats were placed in 80% oxygen immediately after birth and were exposed for either 5, 10, or 14 days, followed by sacrifice or exposure to normoxia for an additional 2, 4, 5, 7, 8, 10, 38, 45 or 56 days. Control rats were simultaneously raised in room air and sacrificed at the same times. All animals were enucleated and their eyes processed for light and electron microscopy. Eyecups were trimmed to facilitate cross-sectioning of the retina in the vertical meridian. No control rats showed signs of retinal hemorrhages or of dysplastic folds or rosettes. Nor did the retinas of rats killed immediately after oxygen exposure contain hemorrhages, but the incidence of retinal folds or rosettes in this group was 54%. For rats exposed to combinations of hyperoxia and brief normoxia (10 days or less), 40% suffered hemorrhages and 50% developed retinal folds or rosettes. Although hemorrhages were more prominent in rats subjected to longer periods of oxygen (73% of all rats exposed for 14 days followed by brief normoxia vs. 6% of those exposed for 5 days followed by brief normoxia), the incidence decreased with time post-exposure in room air. Hemorrhages occurred in 100% of the rats raised in oxygen for 14 days followed by 2 days in room air, and decreased to 50% by 7 days in room air and to 0% by 38 days, indicating a spontaneous resolution with time. In each case, the blood appeared to leak from the newly-forming vessels of the deep capillary net, with most of the red blood cells migrating to the subretinal space. Retinal fold or rosette formation, indicative of developmental dysplasia, occurred in a fraction of virtually all groups of exposed rats, and persisted at the longest post-exposure periods. These two manifestations of oxygen-induced retinopathy are emphasized because they lead to an abnormal separation of the retina from the epithelial layer, which may increase the likelihood of the most serious consequence of ROP--retinal detachment. In fact, all rats that endured post-exposure periods of 38 days or longer before sacrifice exhibited retinal detachment.

Docosahexaenoic acid increases in frog retinal pigment epithelium following rod photoreceptor shedding.

The vertebrate retina conserves docosahexaenoic acid (22:6n-3) during n-3 fatty acid deficiency. The mechanism of conservation is not known, although recycling of this fatty acid between the retinal pigment epithelium (RPE) and retina is one possibility. We examined the role of the RPE in conservation of 22:6n-3 by quantitating the fatty acids and phospholipid molecular species (PLMS) in frog RPE before and after light-stimulated shedding of rod outer segments (ROS). RPE cells were dissociated with brush agitation and purified by a discontinuous ficoll density gradient. One hour after the light-induced shedding of ROS, the phagocytosed ROS tip and opsin content of RPE had increased. Simultaneously, the levels of 22:6n-3 and 22:6(n-3)-containing PLMS were increased in the RPE. Within 8 hr following the shedding event, 22:6n-3 in the RPE had returned to the dark level. These findings indicate that the phagocytosed ROS tips contain 22:6n-3 and that the RPE metabolizes these ROS tips and eliminates 22: 6n-3 from the cell. Thus, the RPE is intimately involved in the metabolism of 22: 6n-3 in the retina. The recycling of 22: 6n-3 from the RPE to the retina is a possible means of conserving this important fatty acid in the retina.

Effect of light history on the rat retina: timecourse of morphological adaptation and readaptation.

Sprague Dawley rats were born and raised under either 5 or 800 lux cyclic light (12L:12D) and were sacrificed at 1, 2, 3, 6, 12, 16, and 28 weeks of age. At each time point outer nuclear layer (ONL) area and rod outer segment (ROS) length were measured. The former is an estimation of photoreceptor number, and the latter is an estimation of the photon-catching integrity of the retina, both of which are known to be dependent on the light environment. Regression analysis revealed an ONL area reduction with time of 0.003 mm2/wk for 5-lux-reared rats and 0.009 mm2/wk for 800-lux-reared rats. ROS length was relatively constant in the dim light group, but showed a decline in 800 lux rats of 0.5 microns/wk. Rats moved from 800 to 5 lux at 9 and 21 wks of age showed no significant change in ONL area after 3 wks. ROS length in these rats increased at a prodigious rate, and in the 12-wk-olds (9 wks at 800 lux, followed by 3 wks at 5 lux), ROS length exceeded that of age-matched rats raised in 5 lux for life.

Conservation of docosahexaenoic acid in the retina.

Over the last several years, evidence has accumulated that n-3 fatty acids, particularly 22:6n-3, are essential for the development of the structure and function of the visual system. The importance of 22:6n-3 is reflected in the tenacious manner in which the retina conserves this fatty acid during n-3 deficiency. We have shown that conservation is achieved by recycling 22:6n-3 within the retina or between the retina and the pigment epithelium. Within the retina, recycling could be accomplished by deacylation-reacylation reactions (Louie et al., 1991; Zimmerman and Keys, 1988). Recycling between the retina and the RPE may be achieved through specific transport proteins, possibly interphotoreceptor retinoid-binding protein (Bazan et al., 1985) and/or apolipoprotein E (Bazan et al., 1991).

Conservation of docosahexaenoic acid in rod outer segments of rat retina during n-3 and n-6 fatty acid deficiency.

We investigated the mechanism by which rat retina conserves docosahexaenoic acid during essential fatty acid deficiency. Weanling female albino rats were fed diets containing either 10% by weight hydrogenated coconut oil, safflower oil, or linseed oil for 15 weeks. Plasma and rod outer segment (ROS) membranes were prepared for fatty acid and phospholipid molecular species analysis. In addition, retinas were removed for morphometric analysis. We found the following: (1) Plasma phospholipids and cholesterol esters from coconut oil, safflower oil, and linseed oil diet groups were enriched in 20:3(n-9), 20:4(n-6), and 20:5(n-3), respectively. The levels of these 20-carbon fatty acids in the ROS, however, were only slightly affected by diet. (2) The fatty acids and molecular species of ROS phospholipids from the safflower oil and coconut oil groups showed a selective replacement of 22:6(n-3) with 22:5(n-6), as evidenced by a reduction of the 22:6(n-3)-22:6(n-3) molecular species and an increase in the 22:5(n-6)-22:6(n-3) species. (3) The renewal rate of ROS integral proteins, determined by autoradiography, was 10% per day for each diet group. (4) Morphometric analysis of retinas showed no differences in the outer nuclear layer area or in ROS length between the three groups. We conclude that the conservation of 22:6(n-3) in ROS is not accomplished through reductions in the rate of membrane turnover, the total amount of ROS membranes, or in the number of rod cells. The retina may conserve 22:6(n-3) through recycling within the retina or between the retina and the pigment epithelium, or through the selective uptake of 22-carbon polyunsaturated fatty acids from the circulation.

Identification and immunolocalization of phospholipase C in bovine rod outer segments.

Bovine rod outer segments (ROS) contain a phospholipase C (PLC) that hydrolyzes phosphatidylinositol 4,5-bisphosphate. Approximately 60-70% of PLC activity is recovered in soluble extracts of ROS. Moreover, the specific activity of this soluble PLC is approximately 10-fold higher than that of resealed ROS enzyme activity. Peptide-specific antiserum (Ab 1109) directed against a highly conserved sequence of the Y-region found in several PLC isozymes was used to detect any PLC belonging to this family. This antibody specifically recognized a protein of apparent molecular mass of approximately 140 kDa present in immunoblots of soluble extracts of both ROS and whole retina. The elution profile of this 140-kDa antigen from a Sephadex G-150 column coincided with the peak of PLC activity, suggesting PLC activity is associated with the 140-kDa protein. Immunocytochemical studies of bovine retina using Ab 1109 showed pronounced immunoreactive labeling in the photoreceptor layer. In resealed ROS and washed ROS membranes, Ab 1109 recognized an additional protein of apparent molecular mass of 70 kDa not usually detectable in soluble extracts of ROS, suggesting the presence of at least two isozymes of PLC in ROS.

Predisposing factors to light-induced photoreceptor cell damage: retinal changes in maturing rats.

Retinal changes occurring during the period of growth and maturation of Long Evans pigmented rats were examined to obtain a better understanding of the basis for the age-dependency of light-induced photoreceptor cell damage. Susceptibility to light damage increased markedly between 30 and 60 days of age and to a lesser extent between 60 and 90 days. Although the retinal antioxidant vitamins E and C, and taurine showed a significant increase during the age-period studied, retinal lipid phosphorus and total protein increased by similar amounts indicating that the concentration of these nutrients was not changing. In contrast, rhodopsin content of the retina increased progressively by 44% between 30 and 90 days of age. While ROS length showed no appreciable change with age, rhodopsin per ROS length increased by 31% between 30 and 60 days of age and by 48% between 30 and 90 days. Determinations of ROS phospholipid to rhodopsin ratio and disks per ROS length indicated that rhodopsin did not become more concentrated in photoreceptor cells between 30 and 90 days. However, the 12% increase in ROS diameter between 30 and 90 days of age may partially account for the rhodopsin difference. These findings demonstrate an age-dependent association between greater rhodopsin per ROS length and increased susceptibility to retinal light damage. An increased metabolic demand on photoreceptor cells with greater rhodopsin may be an important factor influencing their destruction by light.