Stephen H. Sinclair

Altered Retinal Vascular Response to 100% Oxygen Breathing in Diabetes Mellitus

The effect of 100% oxygen breathing on retinal blood flow was investigated using laser Doppler velocimetry in 19 normal eyes, and in 41 eyes of insulin treated diabetic patients. Of the diabetic eyes studied, nine had no retinopathy, 18 had background diabetic retinopathy, seven had proliferative diabetic retinopathy, and seven had proliferative diabetic retinopathy that had been previously treated by argon panretinal photocoagulation. Five minutes of 100% oxygen breathing produced an average decrease in blood flow of 61% (SD = 8) in normal eyes, 53% (SD = 10) in NR eyes, 38% (SD = 13) in background diabetic retinopathy eyes, 24% (SD = 18) in proliferative diabetic retinopathy eyes and 54% (SD = 8) in panretinal photocoagulation eyes. In six eyes with proliferative retinopathy measured before and after panretinal photocoagulation, a significant increase in vascular response to O2 was observed following photocoagulation (Wilcoxon signed rank test, P less than 0.05).

Intravitreal Injection of Erythropoietin Protects both Retinal Vascular and Neuronal Cells in Early Diabetes

PURPOSE To explore and evaluate the protective effect of erythropoietin (EPO) on retinal cells of chemically induced diabetic rats after EPO was injected intravitreally at the onset of diabetes. METHODS Diabetes was induced in Sprague-Dawley rats by intraperitoneal injection of streptozotocin (STZ). At the onset of diabetes, a single intravitreal injection of EPO (0.05-200 ng/eye) was performed. In the following 6 weeks, the blood retinal barrier (BRB) was evaluated by Evans blue permeation (EBP). Retinal cell death in different layers was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. The retinal thickness and cell counts were examined at the light microscopic level. Electron microscopy (EM) was used to scrutinize retinal vascular and neuronal injury. Neurosensory retinas of normal and diabetic rats were used as the sources of reverse transcription-polymerase chain reaction (RT-PCR) and Western blot for the detection of EPO, EPO receptor (EpoR), and products of the extracellular signal-regulated kinase (ERK) and the signal transducers and activators of transcription 5 (STAT5) pathways. The distribution of EpoR in retinal layers was demonstrated by immunohistochemistry (IHC). RESULTS In the diabetic rats, BRB breakdown was detected soon after the onset of diabetes, peaked at 2 weeks, and reached a plateau at 2 to 4 weeks. The number of TUNEL-positive cells increased in the neurosensory retina, especially, the outer nuclear layer (ONL) at 1 week after diabetes onset and reached a peak at 4 to 6 weeks. The retinal thickness and the number of cells in the ONL were reduced significantly. EM observations demonstrated vascular and photoreceptor cell death starting soon after the onset of diabetes. All these changes were largely prevented by EPO treatment. Upregulation of EpoR in the neurosensory retina was detected at both the transcriptional and protein levels 4 to 8 weeks after the onset of diabetes, whereas, the endogenous EPO levels of neurosensory retinas were essentially unchanged during the same period observed. In EPO-treated diabetic groups, EpoR expression remained at upregulated levels. Within 2 weeks of the onset of diabetes, activation of the ERK but not the STAT5 pathway was detected in the diabetic retina treated with EPO. CONCLUSIONS These data demonstrate that apoptosis is an major contributor to neuronal cell death in the early course of diabetic retinopathy (DR). The upregulation of EpoR may be a compensatory response of retinal cells and tissue to diabetic stresses. The EPO/EpoR system as a maintenance-survival mechanism of retinal neurons responds to the insults of early diabetes other than ischemia. The protective function of EPO/EpoR at the least acts through the EpoR-mediated ERK pathway. Exogenous EPO administration by intravitreal injection in early diabetes may prevent retinal cell death and protect the BRB function. Therefore, this is a novel approach for treatment of early DR.

Retinal vascular autoregulation in diabetes mellitus.

The blue field entoptic technique was used to study autoregulation of the macular retinal circulation in response to acute alterations of intraocular pressure in 71 diabetic eyes and 30 normals matched for age, systemic blood pressure, and ophthalmic artery diastolic pressure. IOPmax, the maximal intraocular pressure at which flow is maintained normal by autoregulation, was normal in eyes with no retinopathy (30 +/- 3.2 mm Hg) but decreased with progression of retinopathy, approaching the resting intraocular pressure in eyes with proliferative retinopathy. The hyperemia observed by normals to an acute reduction of intraocular pressure was not observed frequently in the diabetics with no retinopathy. The frequency of observation of the hyperemia decreased with progression of retinopathy and was uniformly absent in eyes with proliferative retinopathy. A group of eyes with minimal microangiopathy was found to have an abnormal IOPmax and no hyperemic response. The prognostic significance of these parameters remains to be established.

Fundus camera based retinal LDV

A new bidirectional laser Doppler velocimeter (LDV) is described for absolute measurement of the speed of red blood cells flowing in individual retinal vessels. The basic component of the instrument is a standard retinal camera that eliminates the need for a contact lens. The laser beam is delivered to the eye through the fundus illumination optical system of the camera. Target fixation is done with the eye under examination. The measurements are independent from the ocular refraction; only the axial length of the eye need be determined. The instrument markedly simplifies the technique of retinal blood flow measurement.

ERK- and Akt-dependent neuroprotection by erythropoietin (EPO) against glyoxal-AGEs via modulation of Bcl-xL, Bax, and BAD.

PURPOSE To characterize the neuroprotective mechanisms of erythropoietin (EPO) against the stress of glyoxal-advanced glycation end products (AGEs) in retinal neuronal cells. METHODS Rat retinal organ culture, primary retinal neuron culture, and retinal cell line (R28 cell) culture under glyoxal-AGEs insult were used as in vitro models. Exogenous EPO was applied to these models. Retinal neuronal cell death was assessed by TUNEL, ethidium bromide/acridine orange staining, and cell viability assay. R28 cell proliferation was evaluated by BrdU incorporation and propidium iodide staining. Real-time RT-PCR and Western blot analysis were used to detect Bcl-xL, Bcl-2, Bax, BAD, and products of extracellular signal regulated kinase (ERK) and Akt pathways. Specific inhibitors and plasmids were used to pinpoint the roles of ERK and Akt pathways. Results. EPO protected the retinal cells from glyoxal-AGE-induced injury in a time- and dose-dependent fashion. The protective function of EPO was proved to be antiapoptotic, not pro-cell proliferative. Glyoxal upregulated Bax expression but suppressed Bcl-xL expression and BAD phosphorylation. In contrast, EPO enhanced BAD phosphorylation and Bcl-xL expression but downregulated Bax. The regulation of these apoptosis-related proteins by EPO was through ERK and Akt pathways. CONCLUSIONS These data demonstrate that exogenous EPO significantly attenuates the retinal neuronal cell death induced by glyoxal-AGEs by promoting antiapoptotic and suppressing apoptotic proteins. EPO/EPO receptor signaling through ERK and Akt pathways is pivotal in EPO neuroprotective mechanisms.

Macular Retinal Capillary Hemodynamics in Diabetic Patients

Macular retinal capillary hemodynamics was evaluated in 39 nonhypertensive insulin-dependent diabetic patients and 24 age-matched control subjects using the blue field entopic simulation technique. A statistically significant 25% increase in macular capillary flow velocity was observed among the diabetic eyes along with a 37% decrease in the density of the entoptically perceived leukocytes. When the eyes of diabetic patients were graded according to the modified composite scale of Klein et al, capillary flow velocity was elevated in the group without retinopathy as well as in those with mild background retinopathy and those with preproliferative or proliferative retinopathy. The density of the entopically perceived leukocytes was more severely reduced in those with retinopathy than in those without retinopathy but was poorly correlated with the composite grading scale. These results are consistent with the concept that in diabetes, capillary obstruction, either transient or permanent, may focally occur within the retina associated with vasodilation in the adjacent microvasculature because of relative tissue hypoxia.

A Long Krupin-Denver Valve Implant Attached to a 180° Scleral Explant for Glaucoma Surgery

Abstract A long glaucoma valve implant attached to an external scleral explant was used during filtration surgery in 72 eyes: 39 eyes with neovascular glaucoma and 33 eyes with other types of secondary glaucomas or with primary glaucoma in which prior filtration surgery had failed. The implant consisted of an open Silastic tube (outside diameter, 0.64 mm), which was placed into the anterior chamber. The external end of the tube contained a pressure-sensitive (opening pressure, 11 mmHg) and unidirectional slit-valve, and was sutured within the groove of a #220 Silastic explant. The 180° explant was placed beneath three rectus muscles and then sutured so that the grooved side was against the sclera, with the anterior edge 8 to 12 mm posterior to the limbus. The long glaucoma valve implant resulted in a large, posterior bleb extending over the area of the Silastic explant. The mean preoperative intraocular pressure (IOP) of 43.9 mmHg in the eyes with neovascular glaucoma was reduced to 17.4 mmHg after a mean follow-up of 20.2 months. The mean preoperative IOP of 38.1 mmHg in the eyes after failure of previous filtration surgery was reduced to 17.6 mmHg at a mean follow-up of 21.0 months. Postoperative IOP was less than 21 mmHg in 77% of eyes with neovascular glaucoma (47% required additional medication) and in 82% of eyes with previous failure of filtration surgery (56% required additional medication).

Effects of intravitreal erythropoietin therapy for patients with chronic and progressive diabetic macular edema.

BACKGROUND AND OBJECTIVE To determine the effects of intravitreal injections of erythropoietin in eyes with severe, chronic diabetic macular edema, 5 eyes of 5 patients underwent injections of rHuEPO alpha (EPO). PATIENTS AND METHODS All eyes had progressive vision loss and persistent or worsening edema with prior multi-modal treatment. EPO (5U/50 microL) was injected intravitreally every 6 weeks for three doses and followed for an additional 6 weeks with complete ocular examinations, fluorescein angiography, optical coherence tomography (OCT), and central field acuity perimetry. RESULTS Visual acuity of all patients was subjectively improved by 3 or more lines in 3 eyes and 1 line in 2 eyes. Visual acuity improved to a larger extent than anatomic improvement by OCT. Clearing of hard exudates but only minor improvement in leakage on fluorescein angiography was observed. Improvement in vision occurred within 1 week after the first injection and was maintained until the end point of the current case series (at 18 weeks after the first injection). CONCLUSION This case series seems to show a short-term positive response to EPO for a specific group of patients with chronic diabetic macular edema who were unresponsive to currently available therapies.

Pharmacokinetic and toxicity study of intravitreal erythropoietin in rabbits

AbstractAim:To study the pharmacokinetics and toxicity of intravitreal erythropoietin (EPO) for potential clinical use.Methods:For toxicity study, 4 groups (60 rabbits) with intravitreal injection (IVit) of EPO were studied (10U, 100U, or 1 000U) per eye for single injection and 0.6 U/eye (the designed therapeutic level in rabbits) for monthly injections (6×). Eye examination, flash electroretinogram (ERG), and fluorescein angiography (FA) were carried out before and after injection. The rabbits were killed for histological study at different intervals. For the pharmacokinetic study, after IVit of 5 U EPO into left eyes, 44 rabbits were killed at different intervals, and the EPO levels in vitreous, aqueous, retina and serum were analyzed by enzyme-linked immunosorbent assay.Results:At all of the time points examined, the eyes were within normal limits. No significant ERG or FA change was observed. The histology of retina remained unchanged. The pharmacokinetic profile of EPO in ocular compartments was summarized as follows. The half-life times of EPO in vitreous, aqueous and serum were 2.84, 3.24 and 2.12 d, respectively; and Cmax were 4615.75, 294.31 and 1.60 U/L, respectively. EPO concentrations in the retina of the injected eye peaked at 1.36 U/g protein at 6 h following injection, with the half-life observed to be 3.42 d.Conclusions:IVit of EPO in a wide range is well tolerated and safe for rabbit eyes. At doses up to 10-fold higher than therapeutic levels, EPO has a pharmacokinetic profile with faster clearance, which is favorable for episodic IVit.

The Retinal Venous Pulse: Its Relation to Optic Disc Characteristics and Choroidal Pulse

PURPOSE The presence or absence of the spontaneous retinal venous pulse is an important clinical sign, especially when one is evaluating a patient who may have increased intracranial pressure. The purpose of this study is to evaluate a large group of healthy patients, correlating optic disc characteristics, choroidal pulse, and brachial pulse pressure with the presence and intensity of the spontaneous retinal venous pulse. METHODS Ninety-seven patients were studied correlating these parameters with their fundus photographs. Ocular pneumotonometry as a measure of the choroidal pulse and the brachial pulse pressure were correlated with the optic disc characteristics and the intensity of the retinal venous pulse statistically. RESULTS The results showed a highly significant correlation with these parameters. The greater the pulse amplitude--especially the choroidal pulse the more likely the spontaneous retinal venous pulse would be present. More importantly, the anatomic variable of optic disc cup and vascular configuration had the greatest influence on whether the spontaneous retinal venous pulse is present or absent. CONCLUSION The authors conclude that unless the clinician is aware of the importance of optic nerve characteristics and pulse amplitude in the choroid (indirectly measured in a normal clinical setting by the brachial pulse) the significance of the spontaneous retinal venous pulse cannot be properly determined.