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Purpose: Demonstrate in vivo that controlled and extended release of a low dose of anti-vascular endothelial growth factor (anti-VEGF) from a microsphere-hydrogel drug delivery system (DDS) has a therapeutic effect in a laser-induced rat model of choroidal neovascularization (CNV)., Methods: Anti-VEGF (ranibizumab or aflibercept) was loaded into poly(lactic-co-glycolic acid) microspheres that were then suspended within an injectable poly(N-isopropylacrylamide)-based thermo-responsive hydrogel DDS.The DDS was shown previously to release bioactive anti-VEGF for ~200 days. CNV was induced using an Ar-green laser. The four experimental groups were as follows: (i) non-treated, (ii) drug-free DDS, (iii) anti-VEGF-loaded DDS, and (iv) bolus injection of anti-VEGF. CNV lesion areas were measured based on fluorescein angiograms and quantified using a multi-Otsu thresholding technique. Intraocular pressure (IOP) and dark-adapted electroretinogram (ERG) were also obtained pre- and post-treatment (1, 2, 4, 8, and 12 weeks)., Results: The anti-VEGF-loaded DDS group had significantly smaller (60%) CNV lesion areas than non-treated animals throughout the study. A small transient increase in IOP was seen immediately after injection; however, all IOP measurements at all time points were within the normal range. There were no significant changes in ERG maximal response compared to pre-treatment measurements for the drug-loaded DDS, which suggests no adverse effects on retinal cellular function., Conclusions: The current study demonstrates that the DDS can effectively decrease laser-induced CNV lesions in a murine model. Controlled and extended release from our DDS achieved greater treatment efficacy using an order of magnitude less drug than what is required with bolus administration. This suggests that our DDS may provide a significant advantage in the treatment of posterior segment eye diseases.

Purpose: To directly measure in vivo retinal nitric oxide (NO) concentration in experimental early diabetic retinopathy and correlate measurements with blood glucose to determine how intraretinal NO changes with severity of diabetes., Methods: Long-Evans rats were made diabetic with streptozotocin (STZ). Three weeks post STZ injection, intraretinal NO concentration profiles were recorded using a dual NO/electroretinogram microelectrode. Diabetic profiles were compared with profiles from healthy controls, healthy rats injected with the NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME), and healthy rats that received acute glucose injections ("acute hyperglycemia"). The NO values at the retina/RPE boundary (100% retinal depth) and retinal surface (0% depth) were analyzed for correlation with blood glucose., Results: The average NO concentrations in the outer retina, inner retina, and vitreous humor of mild diabetic rats (250-400 mg/dL) were significantly higher than controls by 73%, 47%, and 70%, respectively. The average NO concentrations in the outer retina, inner retina, and vitreous humor of severe diabetic rats (500-600 mg/dL) were lower than controls, with NO at 41%, 36%, and 36% of controls, respectively, similar to L-NAME-treated eyes (38%, 36%, 20% of control). The NO levels in moderate diabetic rats (400-500 mg/dL) and acute hyperglycemia rats were similar to controls. The NO was significantly and inversely correlated with blood glucose for diabetic rats at 100% depth (R = -0.91) and 0% depth (R = -0.79) but not for acute hyperglycemia rats., Conclusions: The higher-than-control level of NO in mild diabetic rats and lower-than-control level in severe diabetic rats show that severity of diabetes is an important factor when measuring the bioavailability of NO in diabetic retinopathy., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Nitric oxide (NO) plays an important physiological role in normal and pathological retinas. Intraretinal NO concentrations have not been directly measured due to lack of NO electrodes capable of determining their location in the retina. The microelectrodes described here allow recording of the intraretinal electroretinogram (ERG) and NO concentration from the same location, with ERGs used to determine retinal depth. Double-barreled electrodes were constructed with one barrel serving as a reference/voltage recording barrel and the other containing a Nafion-coated carbon fiber used to detect NO amperometrically. Nafion coating imparted a high selectivity for NO versus ascorbic acid (2000:1). In vivo rodent experiments demonstrated that the electrodes could record intraretinal ERGs and NO current with minimal retinal thickness deformation (9%), allowing for retinal NO depth profile measurements. Comparison of NO depth profiles under control conditions and under nitric oxide synthase (NOS) inhibition by 5 mM L-NG-Nitroarginine methyl ester (L-NAME) verified that the recorded current was attributable to NO. NO concentrations from control profiles ( n = 4) were 2.37 ± 0.34 μM at the choroid and 1.12 ± 0.14 μM at the retinal surface. NO concentrations from L-NAME profiles ( n = 4) were significantly lower at 0.83 ± 0.15 μM at the choroid ( p = 0.006) and 0.27 ± 0.04 μM at the retinal surface ( p = 0.001). Localized regions of increased NO (100-400 nM) were seen in the inner retina under control conditions but not after L-NAME. The dual ERG-NO electrode may be a valuable tool in evaluating the role of NO in normal and diseased retinas.

The purpose of this study was to develop a non-biased method of quantitatively measuring choroidal neovascularization (CNV) areas based on late-phase fluorescein angiography (FA) images. Experimental CNV was induced in Long Evans rats by laser disruption of the Bruch's membrane. FA was performed weekly for 5weeks. Multi-Otsu thresholding (MOT) was used to quantify CNV in late-phase FA images from both experimental rodent CNV and wet age-related macular degeneration (wAMD) patients. Images were automatically thresholded into three levels based on the image histogram, with the highest level containing CNV. To determine the technique's ability to quantify CNV areas, rats were given either triamcinolone acetonide or dexamethasone sodium phosphate to treat CNV and compared to untreated rats. The rat CNV lesion areas measured from 5-week histology sections from each treatment group were compared to areas measured from the corresponding FA images. MOT was able to detect statistical decreases in rodent CNV area in the treatment groups versus control from weeks 3 through 5. The ratio of CNV area measured from histology to area measured from FA images was not statistically different between groups. Finally, to determine the usefulness of MOT on pathological morphologies of CNV, MOT was performed on late-phase FA images from patients with classic and diffuse CNV. The technique was able to segment classical CNV in wAMD patients, but performed poorly with diffuse CNV. MOT provides a robust, objective, and quantifiable area measurement of CNV lesion area in both experimentally-induced and pathological CNV. The results indicate that MOT could be a useful research tool in helping evaluate the effects of therapeutics on CNV growth., (Copyright © 2013 Elsevier Inc. All rights reserved.)

There is significant interest in biomaterials that provide sustained release of therapeutic molecules to the retina. Poly(N-isopropylacrylamide) (PNIPAAm)-based materials have received significant attention as injectable drug delivery platforms due to PNIPAAm's thermo-responsive properties at approximately 32 °C. While the drug delivery properties of PNIPAAm materials have been studied extensively, there is a need to evaluate the safety effects of hydrogel injection on retinal function. The purpose of this study was to examine the effect of poly(ethylene glycol) diacrylate (PEG-DA) crosslinked PNIPAAm hydrogel injection on retinal function. Utilizing scanning laser ophthalmoscopy (SLO), optical coherent tomography (OCT), and electroretinography (ERG), retinal function was assessed following hydrogel injection. In region near the hydrogel, there was a significant decrease in arterial and venous diameters (∼4%) and an increase in venous blood velocity (∼8%) 1 week post-injection. Retinal thickness decreased (∼6%) at 1 week and the maximum a- and b-wave amplitudes of ERG decreased (∼15%). All data returned to baseline values after week 1. These data suggest that the injection of PEG-DA crosslinked PNIPAAm hydrogel results in a small transient effect on retinal function without any long-term effects. These results further support the potential of PNIPAAm-based materials as an ocular drug delivery platform., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

This study evaluated the effect of exogenous PGF(2)alpha on circulating LH concentrations in postpartum multiparous (n = 32) and primiparous (n = 46) Brahman cows. The cows were randomly allotted within parity and calving date to receive 0, 1, 2 or 3 mg im PGF(2)alpha (alfaprostol)/100 kg body weight (BW), with or without GnRH on Day 30 after calving. Blood samples were collected at weekly intervals from calving through treatment. Serum progesterone concentrations were determined using RIA procedures to assure that only anestrous cows were treated. Sterile marker bulls were maintained with cows on Coastal bermudagrass pastures until the first estrus was detected. Multiparous cows had a shorter (P < 0.05) interval from calving to estrus than did primiparous cows. Serum LH was affected by time (P < 0.0001), PGF(2)alpha dose (P < 0.0002), GnRH (P < 0.0001), parity by PGF(2)alpha dose (P < 0.0003), PGF(2)alpha dose by GnRH (P < 0.0009), parity by GnRH (P < 0.0008), and by parity by PGF(2)alpha dose by GnRH (P < 0.0005). Multiparous cows not receiving GnRH had higher mean serum LH (P < 0.02), LH peak pulse height (P < 0.03), and area under the LH release curve (P < 0.03) compared with primiparous cows. The number of LH pulses/6 h was greater (P < 0.06) in multiparous than primiparous cows, and was greater (P < 0.02) in multiparous cows receiving 3 mg/100 kg BW than in cows receiving 2 mg/100 kg BW, but not in the controls or in cows receiving 1 mg/100 kg BW. Exogenous GnRH resulted in increased (P < 0.0001) serum LH concentrations in all cows, and LH was enhanced (P < 0.0009) by simultaneous treatment with PGF(2)alpha. Primiparous cows had a greater response (P < 0.0005) to PGF(2)alpha and GnRH compared with multiparous cows. Pituitary release of LH in response to GnRH was enhanced by simultaneous exposure to PGF(2)alpha in Day 30 postpartum cows.

The effects of ambient temperature and source of exogenous energy (glucose or colostrum) on the ability of newborn Brahman calves to maintain rectal temperature (RT) were determined. All calves were removed from dams within 30 min of birth, before suckling. Calves were catheterized and placed in either a warm (25 degrees C) or cold (5 degrees C) environment for 150 min and given either colostrum or glucose. This resulted in four groups (warm colostrum, n = 7; cold colostrum, n = 7; warm glucose, n = 6; cold glucose, n = 6). Blood samples and RT were obtained at 15-min intervals during warm or cold through 150 min, when calves were removed from cold, and at 180, 240, and 300 min. After 60 min, each calf was given either 1 L of colostrum (38 degrees C) from its dam or glucose (38 degrees C) infusion of 750 mg/kg BW. Plasma glucose concentrations were determined by enzymatic techniques and serum insulin concentrations by RIA. Calves exposed to cold or warm air temperatures had similar declines in rectal temperature from 0 to 60 min. Colostrum-fed, cold calves had a greater (P less than .07) decrease in RT than did colostrum-fed, warm calves from 75 through 150 min; glucose-infused warm and cold calves had intermediate decreases in RT. Plasma glucose increased (P less than .0001) in glucose-infused compared with colostrum-fed calves at 75 min, but glucose-infused calves had lower (P less than .02) glucose levels from 180 to 300 min. Higher (P less than .05) glucose concentrations

1. Low xanthine dehydrogenase (LXD) mutant Drosophila melanogaster were fed 0.2% adenine for 7 generations, no adenine for the next 2 generations (relaxed) and 0.2% adenine again for the next 3 generations (rechallenged) to obtain adenine-resistant lines of Drosophila (LXD-adenine). Flies grown without adenine served as LXD-controls. 2. Purines ranked as follows; adenine > adenosine > AMP > inosine > IMP in decreasing order of toxicity to LXD-adenine flies. 3. Addition of ribose to 9N position, or phosphate or carboxy to 6C position of the purine ring alleviated the toxicity. 4. More LXD-adenine offspring survived than did LXD-control offspring rechallenged with adenine.

Brahman calves (n = 28) were used to evaluate the effect of environmental temperature during the 1st or 2nd d after birth. Calves were removed from their dams within 30 min of birth (newborn; D0) before suckling or at 20 h of age and fasted for 4 h before treatment (day-old; D1). Calves were placed in either a warm (W; 25 degrees C) or a cold (C; 5 degrees C) environment for 2 h and either maintained in or transferred to, respectively, W for 22 h. Blood samples were collected via jugular catheters at 15-min intervals beginning at initial placement in W or C through 3 h and at 4, 5, 6, 8, 10, 12, 14, 18, and 24 h. Rectal temperature (Tr) was recorded with each sample. Following the 60-min and 12-h samples, each calf was administered 1 liter of colostrum from its dam. Serum or plasma was analyzed for glucose, lactate, plasma urea nitrogen, triglycerides, nonesterified fatty acids, insulin, cortisol, triiodothyronine (T3), and thyroxine (T4). Rectal temperature of D0C calves was lower (P less than .05) than that of other calves from 75 min through 3 h. Insulin, lactate, T3, and plasma urea nitrogen concentrations were not different among all calves. Higher (P less than .01) cortisol and T4 concentrations were observed in D0 than in D1 calves. Cortisol (P less than .008) and nonesterified fatty acid (P less than .05) levels were greater in C than in W calves. All D0 calves had lower (P less than .0001) glucose concentrations than D1 calves until the 12-h feeding.(ABSTRACT TRUNCATED AT 250 WORDS)

Brahman (n = 9) and 1/2 Simmental x 1/4 Brahman x 1/4 Hereford (n = 11) calves were utilized to determine the influence of exposure to cold on the physiology of the neonate. All calves were removed from their dams within 20 min of birth and prior to suckling. Calves were assigned randomly within breed to either a warm (W; 31 degrees C) or cold (C; 4 degrees C) environmental treatment group. Jugular blood samples were collected via indwelling catheters at 20-min intervals for 180 min. At 100 to 120 min of sampling, all calves were given 1.2 liters of colostrum from their dams via stomach tube. At 120 min, C calves were placed in the W environment. Calf vigor score (CVS) and rectal temperature were determined at each time blood was collected. Serum or plasma was analyzed for glucose (GLU), lactate (LAC), blood urea nitrogen (BUN), hemoglobin (HEM), triglyceride (TRG), triiodothyronine (T3), thyroxine (T4), insulin (INS), cortisol (CORT) and nonesterified fatty acid (NEFA) concentration. Rectal temperature was lower (P less than .01) in C Brahman than in W Brahman and C or W crossbred calves. Crossbred calves had higher (P less than .01) CVS than Brahman calves. Calves in W had lower (P less than .01) GLU than C calves. Brahman calves had higher GLU, LAC, BUN, TRG, T3, T4 and CORT (P less than .05) than crossbred calves. The C Brahman calves had the highest (P less than .05) TRG, CORT, T3 and T4 of all groups. Concentration of NEFA were higher (P less than .01) in C than in W calves.(ABSTRACT TRUNCATED AT 250 WORDS)

Hereford bulls from Montana (MH; n = 15) and Nebraska (NH; n = 15) and Brahman bulls from Texas (BB; n = 18) were relocated to one of three locations (LOC): Montana (MT), Nebraska (NE) or Texas (TX). All bulls were pubertal at the time of relocation in late May 1984. Semen was collected by electroejaculation within 1 wk after relocation and at 90-d intervals beginning in November 1984 through early February 1986. Bulls were given a GnRH challenge (200 micrograms i.m.) during the same week of semen collections. Bulls also were bled for 8 h at 20-min intervals in the fall of 1984 and the spring and fall of 1985 to determine endogenous concentrations of LH and testosterone. Season affected sperm concentration in all breeds (P less than .05) with decreases during the winter in BB and during the summer in NH and MH bulls. Brahman bulls had lower percentage of live cells (LIVE) than NH and MH bulls did (P less than .0001). Brahman bulls decreased in LIVE during the winter (P less than .001). Area under the LH curve after GnRH was lower (P less than .005) in BB than in MH and NH. Brahman bulls in MT had greater (P less than .02) area under the LH curve and lower (P less than .06) area under the testosterone curve than did BB in TX or NE during the winter. There was no seasonal fluctuation in LH or testosterone response to GnRH in NH or MH bulls at any LOC. Area under the endogenous LH curve was lowest (P less than .04) in BB. Basal endogenous testosterone concentration was greater (P less than .03) in NH than in MH or BB. Area under the endogenous testosterone curve was lower (P less than .03) in MH than in NH or BB. These results indicate that BB exhibit seasonal fluctuations in semen quality. This was not so apparent in semen quality traits of Hereford bulls. There also was a seasonal influence in BB on both endogenous testosterone and GnRH-stimulated LH and testosterone concentrations. Compared with Hereford bulls, Brahman bulls had lower endogenous and GnRH-stimulated concentrations of LH.

A trial was conducted to evaluate the ability of a prostaglandin analog, Luprostiol (LP), to synchronize estrus in Brahman cows and heifers. Animals were injected with either 0, 3.75, 7.5, 15 or 30 mg LP or 500 micrograms cloprostenol (CLP) on d 8 or 9 after estrus (d 0). All concentrations of LP (greater than 0 mg) and CLP caused luteolysis in cows and heifers, as indicated by a decline (P less than .01) in serum progesterone concentration after injection. Animals receiving 0 or 3.75 mg LP had a longer (P less than .04) interval to estrus after injection than did animals in other treatment groups. The proportion of animals exhibiting estrus by 120 h after injection was influenced by dose of LP (P less than .0001; 0, 3.75 mg less than 7.5, 15 and 30 mg and CLP) but not by age. Cows had a lower (P less than .01) progesterone concentration than heifers on d 10, 11 and 12 after LP-induced estrus. Progesterone concentration was lowest (P less than .01) on d 10, 11 and 12 after LP-induced estrus in cows given 15 mg LP or CLP. First-service conception rate was similar between cows and heifers, but it was lower (P less than .01) in animals given 15 or 30 mg LP. Both estrogen and LH concentrations were decreased (P less than .01) at the time of estrus by the 15 and 30 mg of LP. Luprostiol can cause luteolysis and estrous synchrony in Brahman cattle.(ABSTRACT TRUNCATED AT 250 WORDS)