Bruce A. Berkowitz

Measurement of capillary permeability from the Gd enhancement curve: A comparison of bolus and constant infusion injection methods

Dynamic imaging of Gd-DTPA uptake has been used by several groups to characterise the permeability of blood-brain barrier and blood-retina barrier lesions, using both bolus and constant infusion rate injections. However, no consensus on which injection protocol is most efficient has been reached. To address this problem, we extend our Simplified Early Enhancement (SEE) theory, applicable to retinal lesions, to cover infusion injections, and demonstrate its application to published data. The two injection methods are compared using computer simulation. We find that, first, an infusion cannot produce a constant plasma concentration in an acceptable time (although a hybrid injection, consisting of a combined bolus and infusion, is able to do this). Second, at any given time after the start of injection, a bolus achieves a higher tissue concentration, and hence enhancement, than does the same dose given as an infusion. Conversely, a bolus achieves any given tissue concentration in a shorter time than the same dose given as an infusion. Consequently, a bolus uses a smaller dose to achieve a given enhancement at a particular time. Third, if renal function is reduced, the error in calculating the permeability from a particular value of enhancement is lower for the bolus than for the infusion. And last, the SEE method is more accurate for a bolus than for an infusion. We conclude that a bolus is always more efficient than an infusion, as well as being easier to administer, and should always be used in preference to an infusion.

Role of dissolved plasma oxygen in hyperoxia-induced contrast

Currently, hyperoxia is being investigated as a method for producing contrast in magnetic resonance images of the brain, solid tumors, and the eye. However, the underlying physiological mechanisms involved in this type of contrast are still not completely understood. For example, under what conditions would dissolved plasma oxygen contribute to the hyperoxia-induced contrast? Using the eye as a model system, we varied the level of dissolved plasma oxygen and observed different patterns of contrast in the vitreous. The observed contrast changes were consistent with tissue oxygen buffering by hemoglobin at an arterial PO2 of 200 mm Hg and dissolved oxygen offloading at arterial PO2s > 350 mm Hg. These data demonstrate that dissolved plasma oxygen does not become an important contrast mechanism until the arterial oxygen tension exceeds approximately 350 mm Hg. The implication of this result to studies in other organs is discussed.

Electron Microscopic Evidence for the Mechanism of Blood-Retinal Barrier Breakdown in Diabetic Rabbits : Comparison with Magnetic Resonance Imaging

Diabetes leads to a breakdown of the blood-retinal barrier (BRB), which can be demonstrated in experimental models by immunocytochemistry and magnetic resonance imaging (MRI). The present study utilizes these methods to investigate the mechanism of BRB breakdown in diabetic rabbits, a model ideally suited to both procedures. Rabbits were treated with alloxan and examined 2 months, 1 year, and 1.5 years after the development of diabetes to assess BRB breakdown using MRI and immunocytochemical staining for endogenous albumin. Using MRI, an increased incidence of retinal vascular leakage is first evident at 1 year of diabetes. Electron microscopic immunolocalization of albumin suggests that BRB compromise is principally mediated by transendothelial transport of serum proteins in endocytic vesicle-like structures of approximately 0.4-1 micron diameter. Some additional retinal vascular leakage is occasionally demonstrated through the interendothelial cell tight junctions, but only when adjacent vascular endothelial cells show degenerative changes. The similarity of these findings to those previously reported for diabetic humans and rats supports the use of the diabetic rabbit as a model for studying BRB dysfunction. MRI and electron microscopic (EM) immunocytochemistry are complementary methods for evaluating BRB dysfunction. MRI can provide an overall picture of the entire eye without sacrificing the animal. EM immunocytochemistry can provide a more detailed picture of a limited area of interest to gain insight into the mechanisms of extravasation. Together, both methods provide a more complete understanding of BRB breakdown in diabetic rabbits.

A comparative study of the effects of argon and diode laser photocoagulation on retinal oxygenation.

Abstract• Background: The purpose of this study was to compare the effects of diode and argon laser photocoagulation (DLP and ALP) on the preretinal oxygen tension (PO2): (1) directly over photocoagulated retina and (2) in between laser lesions. • Methods: DLP or ALP was applied to avascular rabbit retina to produce grade II lesions. On the day of the oxygen measurement, a droplet of perfluorotributylamine was placed into the preretinal vitreous space over the lasered area of retina and the steady-state PO2 was measured in normoxic animals using19F magnetic resonance spectroscopy. To determine the PO2 directly over laser lesions, small (5μl) droplets were placed over large (≈ 4 mm X 5 mm), confluent areas of treatment (burn area ≈95% of the treated retinal surface area). To determine the PO2 in between laser lesions, a larger (10 −μl) droplet was placed over a field of scatter photocoagulation (burn area ≈ 30% of the treated retinal surface area). The theoretical basis for this approach is discussed. • Results: Untreated eyes had a preretinal PO2 of 22±9 mm Hg (mean ± SD,n=15 eyes). The preretinal PO2 was significantly higher over confluent, 12-day-old ALP or DLP lesions (51+13 mm Hg,n=8 eyes;P<0.01) compared to untreated eyes. However, at that time, DLP lesions had significantly higher PO2 values (60±13 mm Hg,n=4 eyes) than did ALP lesions (42±6 mm Hg,n=4 eyes;P=0.04). The preretinal space in between laser lesions generally showed no significant increase in PO2 (P>0.05) over controls on post-treatment days 1, 5, 14 and 47. The only exception was in the DLP group of eyes, in which a significant increase in PO2 over untreated or ALP-treated eyes occurred on post-treatment day 5 (41±7 mm Hg,n=5 eyes;P=0.01). Over photocoagulation lesions in this study, DLP produced a greater increase in preretinal PO2 compared to control values than did ALP. • Conclusions: These results support the use of DLP as an alternative to ALP for the treatment of retinal vascular diseases in which hypoxia is suspected to play a role.

The effect of partial vitrectomy on blood-ocular barrier function in the rabbit

Purpose. To compare ocular vascular permeability in the rabbit after vitrectomy as assessed by contrast-enhanced magnetic imaging (CE-MRI) and measurements of aqueous and vitreous humor protein concentration. Methods. Partial vitrectomies were performed, irrigating with BSS ® or BSS PLUS ®. Post-operative vascular leakage was determined by CE-MRI following intravenous administration of gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA). Aqueous and vitreous protein concentrations were quantified by standard biochemical assay. ERG evaluations were performed on postoperative days 1, 3, and 7. Results. Using BSS as irrigant, breakdown of the inner blood-retinal barrier (BRB) occurred in 4/7 eyes on post-operative day 1. The rate of Gd-DTPA leakage was significantly greater on postoperative day 1 than that in unoperated, control eyes, but declined ˜50% by day 3. At both time points, outer BRB breakdown was restricted to the sclerotomy wounds. No BRB leakage was detectable in control eyes. Blood-aqueous barrier (BAB) leakage was bilateral on day 1. Significantly greater Gd-DTPA leakage occurred in the operated eye than in the nonsurgical contralateral eye. On day 3, ˜40% bilateral reduction in leakage indicated resolution of BAB leakage. Notably, Gd-DTPA leakage of the BAB and BRB was significantly reduced in the BSS PLUS treated group. In contrast to MRI assessments, protein concentrations of the aqueous and vitreous in the surgical eye showed no detectable differences between BSS and BSS PLUS. Concurrent with the transient loss of ocular barrier function, ERG responses also declined. However, by day 7 greater than 90% recovery was noted in BSS PLUS treated animals but not in the BSS treatment group. Conclusions. CE-MRI is capable of detecting subtle changes in vascular permeability following ocular surgery. Advantages of using BSS PLUS compared to BSS as the irrigating solution can be detected using this technique. BSS PLUS’s protection of barrier function is consistent with a rapid recovery in retinal function not observed in BSS treated eyes.

Visualization of subtle contrast-related intensity changes using temporal correlation

Contrast-enhanced magnetic resonance imaging (MRI) is a promising method for investigating the breakdown of the blood-retinal barrier (BRB). However, subtle intensity changes due to low concentrations of contrast agent can be difficult to detect without observer bias. In this study, we developed a temporal correlation method for detecting these subtle signal intensity changes. The method was evaluated in eyes with chemically induced retinal lesions of known size. A time series of MRI data were collected following i.v. administration of different doses of gadolinium-diethylaminetriaminepentaacetic acid (0.05, 0.1, 0.5 mmol/kg). These time course images were analyzed by temporal correlation to a reference enhancement curve. The reference curve was generated based on a validated theoretical enhancement curve. The temporal correlation method detected signal intensity changes in cases where the changes were too subtle to be visible on a postinjection image or a subtraction image (obtained by subtracting the precontrast image from the final image in the time course set). In addition, assessment of leakage was performed by viewing each image in the set with an eight gray-level palette. Areas of leakage identified in this manner corresponded to those identified by temporal correlation, a finding which supports the validity of the temporal correlation method. These results suggest that temporal correlation may be a time-efficient way to screen large numbers of image data sets using an objective, user-independent criterion.

Nondestructive measurement of retinal glucose transport and consumption in vivo using NMR spectroscopy

Abstract: The cellular events underlying various retinopathies are poorly understood but likely involve perturbation of retinal glucose metabolism. Current methods for assessing this metabolism are destructive, thus limiting longitudinal studies. We hypothesize that following an intravitreous injection, the clearance rate of a glucose analogue will be a nondestructive index of retinal glucose transport and metabolism in vivo. First, radiolabeled glucose analogues were injected into the vitreous. After 40 min, the dominant clearance path was posterior via the retina and was consistent with a facilitated transport mechanism. Next, either [6,6‐2H2]glucose or 3‐deoxy‐3‐fluoro‐d‐glucose was injected into the vitreous of rabbit eyes, and the clearance rate of each analogue was determined over 40 min using, respectively, 2H or 19F NMR. These rates were interpreted as a function of the retinal glucose transport and consumption. From the NMR data, the rate of retinal glucose consumption was ∼16 times slower than the transport of glucose. These data demonstrate that NMR measurements of glucose analogue clearance rate from the vitreous can provide a nondestructive index of retinal glucose transport and consumption in vivo.

Preretinal neovascularization in diabetic retinopathy: A preliminary investigation using contrast-enhanced magnetic resonance imaging

Preretinal neovascularization is a well-described feature of advanced diabetic retinopathy. In this study, contrast-enhanced magnetic resonance imaging was used to examine blood-retinal barrier breakdown associated with preretinal neovascularization in three subjects with proliferative diabetic retinopathy. Using a standard imaging protocol, a varying degree of vitreous enhancement was observed in these eyes. The location and severity of enhancement, judged by visual inspection of the images, corresponded to the fluorescein angiographic and/or clinical appearance of preretinal neovascularization. This result suggests that contrast-enhanced magnetic resonance imaging may prove a reasonable approach to the identification of preretinal neovascularization in eyes with significant media opacities.