VEGF-A165b is an endogenous neuroprotective splice isoform of vascular endothelial growth factor A in vivo and in vitro.

Vascular endothelial growth factor (VEGF) A is generated as two isoform families by alternative RNA splicing, represented by VEGF-A165a and VEGF-A165b. These isoforms have opposing actions on vascular permeability, angiogenesis, and vasodilatation. The proangiogenic VEGF-A165a isoform is neuroprotective in hippocampal, dorsal root ganglia, and retinal neurons, but its propermeability, vasodilatatory, and angiogenic properties limit its therapeutic usefulness. In contrast, a neuroprotective effect of endogenous VEGF-A165b on neurons would be advantageous for neurodegenerative pathologies. Endogenous expression of human and rat VEGF-A165b was detected in hippocampal and cortical neurons. VEGF-A165b formed a significant proportion of total VEGF-A in rat brain. Recombinant human VEGF-A165b exerted neuroprotective effects in response to multiple insults, including glutamatergic excitotoxicity in hippocampal neurons, chemotherapy-induced cytotoxicity of dorsal root ganglion neurons, and retinal ganglion cells (RGCs) in rat retinal ischemia-reperfusion injury in vivo. Neuroprotection was dependent on VEGFR2 and MEK1/2 activation but not on p38 or phosphatidylinositol 3-kinase activation. Recombinant human VEGF-A165b is a neuroprotective agent that effectively protects both peripheral and central neurons in vivo and in vitro through VEGFR2, MEK1/2, and inhibition of caspase-3 induction. VEGF-A165b may be therapeutically useful for pathologies that involve neuronal damage, including hippocampal neurodegeneration, glaucoma diabetic retinopathy, and peripheral neuropathy. The endogenous nature of VEGF-A165b expression suggests that non-isoform-specific inhibition of VEGF-A (for antiangiogenic reasons) may be damaging to retinal and sensory neurons.

Evaluation of intraocular pressure elevation in a modified laser-induced glaucoma rat model.

The main drawbacks of currently described pressure induced glaucoma animal models are, that intraocular pressure (IOP) either rises slowly, leading to a heterogeneous onset of glaucoma in the treated animals or that IOP normalizes before significant damage occurs, necessitating re-treatment. Furthermore, a variable magnitude of IOP increase often results when particles are introduced into the anterior chamber. In order to develop a simple and reproducible rat glaucoma model with sustained IOP elevation after a single treatment we induced occlusion of the chamber angle by anterior chamber paracentesis and subsequent laser coagulation of the limbal area with 35, 40 or 45 laser burns. Right eyes served as controls. IOP was measured three times weekly using TonoLab rebound tonometry in awake animals. After four weeks, retinal tissue was harvested and processed for whole mount preparation. The number of prelabeled, fluorogold-positive retinal ganglion cells (RGCs) was analyzed under a fluorescence microscope. The eyes were further analyzed histologically. Results are expressed as means and standard deviation. Amplitude and duration of the IOP elevation increased with the number of laser burns. Two weeks after 35, 40 or 45 translimbal laser burns the IOP difference between treated and control eye was 7.5 ± 5, 14 ± 8 or 19 ± 9 mmHg, respectively; the RGC density/mm(2) 28 days after treatment was 1488 ± 238 for control eyes (n = 31) and 1514 ± 287 (n = 10), 955 ± 378 (n = 10) or 447 ± 350 (n = 11) for the respective laser groups. Mean IOP of all control eyes over the observation period was 12.4 ± 0.8 mmHg. The chamber angle showed pigment accumulation in the trabecular meshwork of all laser groups and confluent peripheral anterior synechia after 40 and 45 laser burns. Histologic examination of the retina revealed increasing glia activation in a pressure dependant manner. In this study, >91% of laser treated rats developed secondary glaucoma with sustained IOP elevation for at least 2 weeks. The amount of IOP elevation and RGC loss correspond with the number of laser burns applied. This relatively high success rate after a single procedure may constitutes an advantage over established glaucoma models, as this decreases the risk of complications (e.g. corneal decompensation, intraocular bleeding or inflammation) and, thus, improves the outcome.

Effect of ischemia duration on autoantibody response in rats undergoing retinal ischemia-reperfusion.

Both the innate and the adaptive immune systems are involved in the pathogenic processes following ischemia-reperfusion injury. We analyzed the possible correlation between the duration of ischemia and autoantibody diversification in a model of ocular ischemia. Rats were subjected to 30, 45, or 90 min of ischemia, and retinal ganglion cell (RGC) density and antibody reactivity were analyzed via customized protein microarray slides. After ocular ischemia, significant alterations in antibody response were observed, while increasing exposure caused more severe RGC damage. Distinct antibody responses after ischemia were detected; these alterations comprised decreased reactivities against cyclophilin A and glyceraldehyde-3-phosphate dehydrogenase, possibly due to increased binding of circulating antibodies to debris material. Other antibodies, like those against α(5)ß(1)-integrin or ß(2) -adrenergic receptor, were upregulated after ischemia.

Quantification of ischemic damage in the rat retina: a comparative study using evoked potentials, electroretinography, and histology.

PURPOSE: To identify objective criteria to quantify visual function in the rat for developing therapeutic strategies to protect neuronal cells after ischemia. The impact of ocular ischemia on luminance and frequency-modulated contrast vision was compared with the function of outer retinal cells and the number of intact retinal ganglion cells (RGCs). METHOD: Ischemia was induced in Brown-Norway rats by elevating the intraocular pressure to 120 mm Hg for 30, 45, 60, and 90 minutes. Visual function was evaluated by visual evoked potentials (VEPs) in awake, freely moving rats. Retinal function was analyzed with scotopic and photopic electroretinography (ERG). RGCs were quantified in retinal flatmounts after postischemic injection of tracer into the superior colliculus. RESULTS: The response to flicker stimulation in VEP recordings decreased as the ischemic episodes increased. The susceptibility to ischemic damage was more pronounced when potentials were evoked with stimuli at higher frequencies. In ERG recordings, ischemia reduced oscillatory potentials and photopic flicker responses more intensely than scotopic a- and b-waves. In counting the RGCs, the reduced cell density correlated significantly with all electrophysiological parameters. The duration of ischemia with half-maximal inhibitory effect was between 36 and 58 minutes for VEPs and between 36 and 41 minutes for ERG, and it was 51 minutes for RGCs. CONCLUSIONS: The amounts of reduction in VEPs, ERG, and RGCs differed as the duration of ischemia increased. The electrophysiological parameters presented in this study may serve as a useful addition to morphologic evaluations in future neuroprotection studies in vivo.

Effects of an extreme endurance race on energy balance and body composition - a case study.

The aim of this case study was to examine energy expenditure (EE) in one cyclist during an extreme endurance cycling race - the "XXAlps 2004" (2,272 km distance and 55,455 m altitude) which was completed in 5 days and 7 hours - and whether the energy deficit derives primarily from the degradation of subcutaneous adipose tissue or loss of muscle mass. Energy intake (EI) was continuously recorded. EE was estimated using two different methods: 1) Continuous heart rate recording using a portable heart rate monitor (POLAR(®) S710) and 2) using the individual relationship between heart rate and oxygen uptake (VO2) determined under laboratory conditions. Body composition was assessed by measuring body mass, skinfold thickness and extremity circumferences. The cyclist lost 2.0 kg body mass, corresponding to 11,950 kcal (50 MJ). Fat mass was reduced by 790 g (7,110 kcal; 30 MJ) and fat free mass by 1.21 kg (4,840 kcal; 20 MJ). Circumferences of the lower extremities were reduced, in contrast skinfold thickness at the lower limbs increased. Energy deficit (ED) was calculated as the difference between EI and EE. Energy deficit using continuous heart rate monitoring was 29,554 kcal (124 MJ), and using the individual relationship between heart rate and VO2 was 7,111 kcal (30 MJ). The results show that the difference between ED due to decreased body mass and ED estimated from continuous heart rate monitoring was 74 MJ (124 MJ - 50 MJ). In contrast the difference between ED due to decreased body mass and ED estimated from laboratory data was 20 MJ (30 - 50 MJ). This difference between methodologies cannot properly be explained. Body mass and skinfold thickness may be overestimated due to hypoproteinemic oedemas during endurance exercise. Data from the present study suggests the individual relationship between heart rate and VO2 may provide a closer estimation of EE during extreme endurance exercise compared with corresponding data derived from continuous heart rate monitoring using the POLAR(®) S710. Key PointsDuring an extreme endurance cycling race, energy expenditure can not be covered by energy intake and an energy deficit results.The energy deficit seems to be covered by degradation of subcutaneous adipose tissue and muscle mass.Determination of energy expenditure during extreme endurance may be properly determined with the individual correlation of heart rate - VO2 instead of continuous heart rate monitoring.

Pattern electroretinogram in glaucoma suspects: new findings from a longitudinal study.

PURPOSE: Early detection of glaucoma remains a challenging problem and needs long-term, prospective studies. The pattern electroretinogram (PERG) directly reflects retinal ganglion cell function. The PERG was evaluated by extending a prospective study of patients with ocular hypertension and evaluated amplitude, PERG ratio, peak time, and trends thereof. METHODS: One hundred twenty eyes of 64 patients with intraocular pressure greater than 25 mm Hg (or ≥ 23 mm Hg with additional risk factors), normal visual fields, normal optic disc appearance, and visual acuity ≥ 0.8 were included in the study. Mean follow-up time was 10.3 years. The per-visit measures of amplitude at 15 reversals/s to 0.8° check size, PERG ratio (0.8°/16°), peak time, visual field, and their trends were analyzed. RESULTS: Over the course of the study 13 eyes converted to glaucoma according to a visual field definition. Amplitude to 0.8° check size, PERG ratio, and peak time were significantly lower in converters. Amplitude and PERG ratio predicted conversion 4 years ahead with a sensitivity/specificity of 67%/64% and 75%/76%, respectively. At this time, the ROC area was already significantly above chance for the PERG ratio. Comparison of the trends of converters and nonconverters revealed significant differences in the PERG ratio; however, trends did not predict conversion as successfully as single-visit measures. CONCLUSIONS: The PERG, especially the PERG ratio, detected glaucoma patients 4 years before visual field changes occurred, with a sensitivity/specificity of 75%/76%. Slope analysis required multiple visits, but provided little additional information in detecting converters.

Face-to-face upright seated positioning for cataract surgery in patients who cannot lie flat.

We describe a technique for cataract surgery in patients unable to adopt the conventional face-to-ceiling position. A standard reclining operating chair and operating microscope are used. Patients are seated instead of lying down, with the chair back elevated 30 to 80 degrees above the horizontal and the operating microscope rotated 45 to 60 degrees to vertical. The surgeon is seated or standing, facing the patient. A clear corneal incision is used via an inferior, temporal, or inferotemporal approach under topical intracameral anesthesia without sedation. Results of this technique in a case series comprising 32 eyes are reported. The technique is useful for patients unable to adopt the traditional approach for cataract surgery and at high risk for complications from general anesthesia. It is technically challenging so should be attempted by experienced surgeons only.

Comparison of intraocular tonometry using three different non-invasive tonometers in children.

BACKGROUND: In childhood glaucoma, the correct determination of intraocular pressure (IOP) is crucial in clinical decision-making. We therefore investigated how intraocular tonometry readings correlate with readings from commonly used tonometers. METHODS: IOP was measured unilaterally in 20 children suffering from congenital (n = 7) or secondary glaucoma (n = 13), 10 minutes after the induction of general anaesthesia. The children were aged from 1 month to 17 years (mean age 4.3 years, median age 1.3 years). Non-invasive applanation (Tono-Pen XL, Perkins tonometer) and indentation tonometry (Schiötz tonometer) were performed in random order prior to intraocular tonometry with a 26-gauge needle connected to a pressure sensor. Linear regression analysis and the coefficients of variance (CV) were used to compare the data obtained from the various tonometers. RESULTS: Compared with intraocular pressure, the CV was 10% for the Tono-Pen XL, 17% for the Schiötz, and 19% for the Perkins tonometer. The coefficient of determination (r(2)) was 0.74 for Tono-Pen XL, 0.60 for Schiötz and 0.78 for Perkins tonometry. The IOP values obtained with the Tono-Pen XL scattered homogeneously around the intraocular IOP, while the Perkins and Schiötz tonometers underestimated intraocular measured IOP. CONCLUSION: Of the three tonometers evaluated in this series of paediatric glaucoma patients, the Tono-Pen XL most closely reflected true IOP.

Methylprednisolone fails to preserve retinal ganglion cells and visual function after ocular ischemia in rats.

PURPOSE: Methylprednisolone (MP) is commonly used to treat traumatic optic neuropathy and optic neuritis, but its benefit in terms of neuronal survival remains controversial. The aim of this study was to investigate the effects of MP on retinal ganglion cell (RGC) survival and visual function after ischemia in rats. METHODS: Ocular ischemia was induced by elevated intraocular pressure. Rats were treated with NaCl, 1 mg/kg/d, or 30 mg/kg/d intraperitoneal MP over 3 days. RGCs were labeled retrogradely 4 days after ischemia and were quantified 6 days later. Post-ischemic retinal function was assessed by scotopic and photopic electroretinography (ERG). Optic nerve function was investigated on days 4 and 10 after ischemia by visual evoked potentials (VEPs). RESULTS: Compared with nonischemic eyes, ischemia reduced RGCs with NaCl to 47% +/- 3% (mean +/- SEM) and to 46% +/- 3% and 43% +/- 6% with 1 mg/kg/d and 30 mg/kg/d MP. ERG did not differ significantly for any parameter among the three groups. Four days after ischemia, the VEPs of rats receiving any dose of MP were significantly higher than the control. VEPs in both steroid groups fell to control levels 10 days after ischemia. CONCLUSIONS: Treatment with MP did not improve RGC survival or retinal function. The VEP showed a short-term benefit because of steroids.