Research
Ronald M. Burde, M.D.
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We are currently continuing our work on a large retrospective review of patients with anterior ischemic optic neuropathy (AION) including disc photographs. Patients with anterior ischemic optic neuropathy are being studied for the presence or absence of drusen within the disc substance. We are using our files as well as those from the Wilmer Eye Institute at Wills Eye Hospital.
It is our expectation that there will be a decreased incidence of anterior ischemic optic neuropathy in patients who have drusen of the optic nerve head as progressive asymptomatic atrophy occurs in these patients. We believe the disc will be decompressed by this atrophy lowering the incidence of anterior ischemic optic neuropathy of the acute variety.
Thus patients with the "disc at risk" would be at increased risk for the development of anterior ischemic optic neuropathy when compared to the general population, but the incidence would be less than that found in patients with the disc at risk without drusen.
If we are able to quantitate the amount of optic atrophy needed to decompress the optic canal in such patients, there would be reason to offer a controlled trial of prophylactic panretinal photocoagulation using xenon arc in order to destroy full thickness retina and produce decompression of this optic nerve head. -
Drs. Slamovits and Burde continue with their interest in the construction of the third nerve nucleus. With the evolution of neuroimaging, it has been possible in-vivo to demonstrate the validity of postulates invoking fasicular lesions as being causative in partial ocular motor palsies in isolation or associated with other neurologic dysfunction. Using cases of our own as well as information available in literature and new hypothetical three dimensional somatotopic constructive of third nerve nucleus and its parasympathetic enlage has been created. This work will be compared with primate material to confirm the dimensions of the third nerve nucleus in an experimental animal.
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Currently Dr. Burde has joined a team of micromolecular geneticists that has developed a method of producing transgenic mice which have the propensity to develop cataracts or retinal tumors. We are studying the evolution of these entities. The development of this group will provide for the opportunity to perform translational research, i.e., applying laboratory techniques to human disease. As mentioned previously, we have just recruited a scientist with expertise in molecular genetics whose major interest is lens.
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We have been investigating the utility of various immunologic parameters in determining the risk for temporal arteritis in patients presenting with acute visual loss. We performed a retrospective study of 56 consecutive patients with suspected temporal arteritis who underwent temporal artery biopsy. The following indices are being evaluated: a complete white blood count, hemoglobin, hematocrit, mean cell volume, platelet count, and erythrocyte sedimentation rate. We are adding C reactive protein. We have been clearly able to demonstrate that platelet count evaluations are specific and useful in the setting of suspected temporal arteritis. The platelet count of greater than 375,000 is compatible with the diagnosis of giant cell arteritis whereas a platelet count of less than 375,000 is unlikely to be found in a patient with giant cell arteritis.
Accidental rupture of the vitreous face with incarceration of vitreous into the surgical wound is a major cause of decreased visual acuity following cataract surgery. Vitreous loss increases the risk of cystoid macular edema, retinal detachment and pupil deformity.
A comparison of various techniques of vitreous loss management in resident cataract surgery was undertaken. A consecutive series of 108 cataract procedures with vitreous loss among 18 residents over a 27 month period was studied. Vitrectomy with methylcellulose sponges in group 1, anterior vitrectomy performed through the original scleral wound in group 2, and anterior and pars plana vitrectomy using a two part system in group 3, were evaluated.
Out of 108 cataract procedures with vitreous loss, 56% occurred with phacoemulsification techniques. 2% of 108 patients had sponge clean up, 79% underwent anterior vitrectomy and 18% had pars plana vitrectomy. 58% of patients had sufficient capsular support for posterior chamber lens insertion and 37% had anterior chamber lens implant. Visual outcome of 20/40 or better can be achieved with meticulous anterior or pars plana vitrectomy following anterior vitreous loss during cataract resident surgery.
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Dr. Fleischman is in the final stages of completing his IRB application for a multicentered clinical trial entitled "Titration Retinal Photocoagulation for Diabetic Neovascularization of the Disc". Since the original Diabetic Retinopathy Study in the late 1970’s very little has changed in the method of treating disc neovascularization by laser. Since that time, however, possible new endpoints for treatment success have been discovered which may prove to alter the amount and therefore the morbidity of laser photocoagulation required to treat this disorder.
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A prototype, inexpensive and portable device has been designed with the purpose to allow diabetic patients, despite 20/20 or near normal vision to test themselves for the presence of clinically significant macular edema. Hopefully, if the specificity and sensitivity of this device pans out, diabetic patients will present earlier to their ophthalmologist by the use of this device. A limited pilot study will be conducted after appropriate IRB permission is granted.
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Teaching Techniques
The development of a low cost television microscope recording methodology using readily available component parts that is also computer interactive is beginning to bear fruit. The first products are currently being used in our institution for teaching rounds. -
Conventional retrobulbar alcohol injection for pain control in blind painful eyes is associated with a significant morbidity. A study was undertaken to determine the efficacy and side effects accompanying the use of retrobulbar chlorpromazine in achieving pain control. 28 consecutive patients with blind painful eyes were studied. Of those receiving retrobulbar chlorpromazine, 82% had a high or moderate degree of pain relief. The mechanism of pain control appears to be independent of a reduction of intraocular pressure. We recommend further case controlled studies to compare chlorpromazine in alcohol and safety efficacy.
Dr. Abramson joined the department at the beginning of the last academic year. Her expertise is in refractive surgery. She is currently using early data from the Bronx to look at a distribution of the types of diseases that are seen and the effectiveness in different populations of photoablative surgery.
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In August of 1993, Dr. Gurland began a review of all strabismus cases performed at Montefiore Hospital and the intent was to compare the surgical results of those who were neurologically impaired compared to those who were normal.
The outcome was the dismaying discovery that a remarkable number of patients in all groups were lost to follow up care following surgery. When follow-up was defined as six months of continuous post-op care, the results revealed that 52% of the neurologically impaired children were lost and 31 out of 67 or 46% of normal patients were also lost to follow-up. This has lead to an attempt with Social Service to attempt to increase compliance and follow-up. -
Dr. Gurland has undertaken a study to characterize the ocular findings in children with perinatally acquired AIDS. 26 of the patients examined had normal ocular examinations while 14 patients had only refractive problems. There were 56 diagnoses; of the remaining 28 patients refractive error was the most common diagnosis followed by strabismus, amblyopia and nystagmus. CMV retinitis was seen in one patient. 18 patients or 26.5% had 28 monocular diagnoses, the most common being asthma.
The ultimate outcome revealed that children who were studied had a greater prevalence of a normal spectrum of ocular pathology than might have been expected. -
Dr. Gurland is continuing epidemiologic studies which are investigating
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the tracking of laboratory results that have been ordered,
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the frequency and effectiveness of refractions for children in the pediatric eye clinics,
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measurements of intraocular tensions in the glaucoma clinic.
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A number of clinical projects being initiated are in the middle of data acquisition.
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Current treatment of infections within the eye has often been an unsatisfying experience for both the patient and physician. The poor ocular penetration of many of the older antibiotics when given intravenously or orally lead to the current practice of performing surgical vitrectomy and injecting antibiotics directly into the eye.
Although we believe that our outcomes have improved, providing an effective and safe level of antimicrobial drug over a sufficient period of time remains illusive. Initial antibiotic concentrations can easily be determined, but the important pharmacokinetic perameters describing the time course of antibiotics in the eye following direct injection or systemic administration are unknown.
Subsequently, management has evolved into an art because there is no scientific paradigm for maintaining therapeutic levels. All other investigators studying intraocular pharmacokinetics have used what is referred to in the literature as "naive pooled data technique". Each subject is sampled only once. A different subject represents each different time point.
Due to the potential for significant intersubject variation extremely large numbers of subjects are required to ensure statistical significance. The reason only one sample was taken per subject was that available assays required large volumes depleting the vitreous cavity of it’s volume. This in itself has been shown to significantly alter the pharmacokinetics.
In our laboratory, an elegant model that allows each subject to have serial samples drawn over the entire time course of an experiment has been developed. This is possible because of a development of a microbiologic assay that only requires 5 microliters of fluid, i.e., literally a drop of vitreous. We have further shown that serial sampling of small volumes of aqueous or vitreous does not affect ocular pharmacokinetics of the antibiotic tested.
We obtained statistically significant data using only a fraction of the subjects that would be required had we used only one specimen per subject. We are using our model to study the effects of different physicochemical properties on the translocation of quinolones, a class of antibacterials and azoles, a class of antifungals into the vitreous humor. Both uninfected and infected models are being studied. Using sophisticated statistical modeling we are now looking at ways by which we will be able to predict intraocular antibiotic levels and improve the outcome of patients with sight-threatening intraocular infections. -
A second collaborative project deals with a tissue culture technique for growing and studying Toxoplasma gondii. Toxoplasmic chorioretinitis is a major cause of posterior uveitis and can cause severe visual impairment and blindness. Our tissue culture technique will allow us to study the disease process and possible treatment modalities. We will be able to examine the influence of different genes on the course of the disease.
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Subconjunctival antibiotics are often administered post-operatively for prophylaxis against bacterial endophthalmitis. Within an institution, variance choice amongst attendings appears to be based on subspecialty. A common prophylactic regime is the combination of an aminoglycoside and acephlosporin favored for synergy providing a broad spectrum of coverage. However, in-vitro inactivation of beta-lactum aminoglycosides is well documented.
A large study was conducted to assess the patterns of post-operative subconjunctival antibiotic use among attending physicians at the Montefiore Medical Center. It was demonstrated that subconjunctival injection of gentamicin and cefazolin were administered 35.6% of the time, cefazolin 32.7%, and vancomycin 9.6%. There was a 40% probability of combined gentamicin and cefazolin use after cataract procedures compared to 14.7% after retinal procedures. Retinal surgery patients had a 47% probability of receiving cefazolin alone compared to 34% for cataract patients.
It is of interest that these results suggest a lack of uniformity in the prophylactic treatment of patients undergoing surgery. The use of cefazolin with gentamicin is the most common post-operative regime used in medical center operating rooms, but because drug inactivation might occur when administered simultaneously, we recommend further studies to delineate the extent of mutual inactivation of aminoglycicides and cephalosporins. At the moment, it would seem that an appropriate prospective study should be undertaken wherein all clinical as well as laboratory data is obtained.
Visual loss secondary to optic nerve ischemia is a common clinical problem. A prototype of chronic ischemia are the open-angle glaucomas. Such ischemia may occur as the result of an acute vascular occlusion or as a chronic phenomenon that may or may not be related to the absolute value of intraocular pressure, e.g., the concentration of NO2 or endothelium.
The long range goals are to understand the underlying mechanisms of optic nerve head ischemia and to eventually develop strategies aimed at protecting the optic nerve head from these processes. The pathophysiology of optic nerve head ischemia is complex, and at the present time it would appear to involve a balance between the factors released by the capillary endothelium, in particular endothelin and nitrous oxide.
Ultimately, cell death appears to be related to the release of excitatory amino acids binding with NMD aspartate - kanate receptors with the production of potentially damaging free radicals. Based on previous work performed in the department, it appears that we should focus on the role of apoptosis (program cell death) in retinal ischemia and its possible relationship with the production of free radicals.
The work would be carried out on an in-vitro model of optic nerve ischemia with subsequent attempts to develop a clinically relevant animal model in which to test the hypothesis that:
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Apoptosis plays a role in the cascade of events leading to optic nerve head damage and axial degeneration accompanying chronic ischemia.
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In-vitro hypoxia, hypoglycemia or a combination of the two induce apoptosis as defined by established morphologic, biochemical and immunohistochemical parameters.
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In-vivo ischemia followed by reperfusion induced apoptosis as determined by established morphologic, biochemical and immunohistochemical parameters.
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Mechanisms that mediate free radicals damage and apoptosis converge in the inner layers of the retina and at the level of the optic nerve head. Extracellular free radical formation leads to rapid cell death by inducing necrosis. In contrast, intracellular free radical formation results in delayed cell death by inducing apoptosis.
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Ganglion cell injury may be prevented or attenuated by agents that prohibit or inhibit apoptosis.
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Endonuclease inhibitors and agents that inhibit macromolecular synthesis afford protection to in-vitro and in-vivo models of retinal ischemia.
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Neurotrophic factors protect the ganglion cells from ischemic damage by inhibiting apoptosis.
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Of interest, it has been recently recognized that many adult cells retain the capacity to undergo apoptosis and that the pathways leading to apoptosis can be activated by a wide array of stimuli including radiation, growth factor withdrawal, treatment with calcium ionophores, and chemotherapeutic agents. Program cell death can also be activated or suppressed by signals from other cells. One of the signals may be the calcium milieu, i.e., changes in intracellular calcium seem to play a role in apoptosis. Calcium, acting as a second messenger, may lead to neurotransmitter release, induction of early genes, activation of protein kinases, activation of endonucleases and activation or inactivation of calcium ions. The laboratories of Dr. Rosenbaum are working on the delineation of this particular cascade.
The following studies are in the process of being performed on the Glaucoma Service:
- The effect of pilocarpine on contrast sensitivity in glaucoma patients. Glaucomatous chronic progressive optic neuropathy causes diffuse psycho-physiologic defects, e.g. field loss and changes in contrast sensitivity. Pilocarpine, an anti-glaucoma agent (parasympathometic agent), often produces severe visual dysfunction in patients - despite the fact that the central visual acuity may not be altered. In the current study, we are looking at the effect of pilocarpine on contrast sensitivity in patients with open angle glaucoma to assess to what extent visual dysfunction occurs in this group.
- Indications for glaucoma surgery. A questionnaire was sent out to the membership of the Glaucoma Society. The purpose of this study is to understand what patterns are presently being used in the decision making process for glaucoma surgery. In précis, we found that most trabeculectomies were performed based on elevated intraocular pressure rather than on progressive cupping or visual field change. Of those surgeries done, based on progressive cupping, the most common method of disc analysis was photography. Longitudinal studies using similar criteria may be useful to further identify appropriate indications for trabeculectomy.
- The effect of PRK on visual fields. Three year data from the Summit PRK Trial is being analyzed with respect to the effect on screening visual fields.
Giant cell arteritis first described in the Takirat of Ali Ibn Isa of Baghdad is an inflammatory disease of unknown origin which leaves affected untreated patients blind in about 40% of cases. The incidence of this disease in Olmstead County is one in a hundred over age 80. The only effective therapy is the use of systemiccorticosteroids for a prolonged period of time and this is associated with a high complication rate including multiple vertebral fractures in 25% of such patients.
Recently methotrexate has been demonstrated to be effective in treating a broad spectrum of inflammatory diseases. A multi-institutional pilot study is being instituted to see if methotrexate can act as a steroid sparing agent, thus reducing the incidence of side effects. In addition, we are attempting to determine if the absolute platelet count can serve as a more accurate prediction of giant cell arteritis than the erythrocyte sedimentation rate.
Our long-term aims are to elucidate the molecular mechanisms mediating both excitation and adaptation within invertebrate photoreceptors, with particular emphasis on mechanisms that may be found more generally in other secretory and sensory transduction systems. In particular, cGMP has been proposed to be the intracellular messenger that gates the light-induced conductance in invertebrate photoreceptors. We have continued our studies on this hypothesis during the past year as detailed below.
Methods: Squid, Loligo pealei, were obtained at the Marine Biological Laboratory, Woods Hole, MA and were dark-adapted in oxygenated sea water for not less than 60 min. Dark-adapted retinas were dissected in dim light. Half retinas were rinsed briefly in sea water with no added calcium ions and frozen by immersion in liquid nitrogen. All subsequent preparative procedures were done at 4o C. A half retina was homogenized in 300 mM homogenization buffer (40 mM Tris-HC1, 5 mM MgC12, 1 mM AEBSF, 4 mM mercaptoethanol, and 0.025 mg/ml leupeptin and 0.025 mg/ml aprotinin, pH = 7.4 adjusted with KOH). For some experiments, 20mM M GTP-S S was added to the homogenization buffer and all subsequent solutions. The homogenate was centrifuged at 35,000 x g for 10 min and the supernatant was saved as the soluble fraction. The pellet was resuspended in 400 homogenization buffer, layered onto 40% sucrose in homogenization buffer, and centrifuged at 35,000 x g for 60 min. The mat of photoreceptor membranes was removed from the interface, resuspended in homogenization buffer, vortexed and centrifuged for 10 min at 35,000 x g. The pellet of photoreceptor membranes was resuspended in homogenization buffer. The photoreceptor membranes were washed 2x with hypotonic buffer (0.5 mM Tris-HC1, 0.5 mM HEPES), 1 mM EDTA, pH = 7.4 adjusted with KOH) and resuspended in homogenization buffer (washed membrane fraction).
Phosphodiesterase activity was assayed continuously by a fluorescence technique using a fluorescent analogue of cGMP, 2’-o-(N-methylanthraniloyl)-3’-5’-cyclic guanosine monophosphate whose fluorescence decreases by as much as 45% when the compound is hydrolyzed. The fluorescence was measured in a fluorometer. Samples were diluted with 40 mM Tris-HC1 plus 5 mM MgC12 at pH = 7.4 in a quartz cuvette (3mm x 3mm) at 20 oC, a Ca-EGTA buffer was added or not, and Mant-cGMP was added.
Results: Membrane fractions of homogenized squid retinas fluoresced (excitation wavelength 280 nm; emission > 520 nm) without the addition of the Mant-cGMP. Therefore, a record of the native membrane fluorescence was subtracted from that recorded with Mant-cGMP present; the difference is a time dependent loss of fluorescence that can be attributed to the fluorescence of the Mant-cGMP. The time dependent loss of Mant-cGMP fluorescence is inhibited by 0.1 mM IBMX. Thus, the time dependent loss of fluorescence can be attributed to the activity of a PDE, that is, the slope of the curve indicates the PDE activity.
PDE activity from membrane and soluble fractions from frozen retinas was also inhibited by addition of EGTA in the presence of 5 mM Mg2+. PDE activity was restored progressively by raising free Ca2+ by the use of Ca-EGTA buffers; free Ca2+ concentrations of 5x10-5 to 10-4 M or greater increased PDE activity to control levels or higher. For both photoreceptor membranes and soluble fractions prepared from frozen retinas, it is clear that increasing Ca2+ increased PDE activity.
It has been demonstrated by several techniques that the intracellular concentration of Ca2+ increases in invertebrate photoreceptors after they have been illuminated. Thus, if modulation by Ca2+ was the dominant control of the aggregate PDE activity, the PDE activity might be hypothesized to increase after illumination of intact photoreceptors. We note that all our measurements are made in the steady-state; we have no information about the time course of the change in PDE activity on the time-scale of the electrical response of the photoreceptor. Nevertheless, our findings suggest that a light-induced increase in Ca2+ may not drive light-elicited down-regulation of cGMP-phosphodiesterase in squid photoreceptors.
Dr. Nawy's laboratory continues to study the biophysical properties of synaptic transmission in the retina. The long-term goal is to relate synaptic transmission at the level of the ion channel to retinal function. Currently he is studying the excitatory amino receptor mediating synaptic transmission from photoreceptors to depolarizing bipolar cells (DPC).
One question that is being addressed is what is the sequence of intracellular events that links the binding of transmitter to the post-synaptic receptor on the DPC to the subsequent opening of nearby synaptic channels. Using the whole cell variation of patch-clamp recording, he has obtained evidence that the glutamate receptors is coupled to a g-protein, which in turn is linked to a cGMP-phosphodiesterase (PDE). When activated, the PDE hydrolyzes cGMP, the substance which is believed to be the activator of the synaptic channel. According to Dr. Nawy's hypothesis, when the concentration of cGMP is reduced by hydrolysis, the channels close. This model is particularly intriguing since it suggests that DPCs utilize the same intercellular cascade as is used by the neighboring photoreceptors for phototransduction.
Over the ensuing year, he will test the hypothesis that cGMP is a second messenger which mediates synaptic transmission in DBCs. Cyclic GMP and other intracellular messenger candidates will be applied to the cytoplasmic surface of excise patches of membrane from DBCs in slices of titer salamander retina.
If cGMP is the intracellular messenger, then we would expect to see electrical responses corresponding to the opening of single channels or ensembles of channels within the patch. Excised patches will be used to study other aspects of these channels, such as their kinetics and ion permeability. These experiments will increase our understanding of the synapse between photoreceptors and DBCs, the first synapse in the visual system.
