Steven T. Schuschereba

Liver and Kidney Injury After Administration of Hemoglobin Cross-Linked with Bis(3,5-Dibromosalicyl) Fumarate

Human hemoglobin cross-linked between the alpha chains with bis (3,5-dibromosalicyl) fumarate (DBBF-Hb) was exchange transfused in swine and the histomorphologic changes were evaluated. Following exchange, animals were euthanized and tissues were taken for light and electron microscopy at 7.5 hours and days 1, 4, 7, and 15. Consistent hepatocellular and renal epithelial cell changes were seen. Hepatic injury, evident at 7.5 hours as cellular vacuolization, progressed to necrosis and acute inflammatory cell infiltration by days 1 and 4, was resolving by 7 days and was completely resolved by day 15. Cytochemical stains for iron and hemoglobin revealed positive material in Kupffer cells, endothelial cells, and necrotic hepatocytes. Rabbit anti-human hemoglobin antibody staining revealed immunoreactive material diffusely present at days 1 and 4 and limited to solitary hepatocytes by day 15. Kidney injury began as proximal tubular epithelial vacuolization and intraluminal casts progressing to tubular necrosis by 24 hours, with resolution by day 15. Iron and hemoglobin stains demonstrated these materials in the early lesions. Immunocytochemistries demonstrated human hemoglobin that remained as late as day 15. Electron microscopy revealed degeneration and regeneration of epithelial cells. The renal lesions were consistent with hemoglobinuria. The liver lesion was less well defined but was self limited.

Laser-induced macular holes demonstrate impaired choroidal perfusion.

Purpose To evaluate choroidal perfusion following creation of a laser-induced macular hole in a nonhuman primate model. Methods Six rhesus monkeys underwent macular exposures delivered by a Q-switched Nd:YAG laser. The lesions were evaluated with fluorescein angiography and indocyanine green angiography using scanning laser ophthalmoscopy. Results Each lesion produced vitreous hemorrhage and progressed to a full-thickness macular hole. Indocyanine green angiography revealed no perfusion of the choriocapillaris beneath the lesion centers. Fluorescein angiography demonstrated mild enlargement of the foveal avascular zone due to loss of perifoveal capillaries. Histopathologic evaluation showed replacement of the choriocapillaris with fibroblasts and connective tissue. Conclusions Nd:YAG laser–induced macular holes result in long-term impairment of choroidal perfusion at the base of the hole due to choroidal scarring and obliteration of the choriocapillaris. Evaluation of choroidal perfusion may be useful in assessment of laser-injured patients. Impairment in choroidal perfusion may have functional implications for surviving photoreceptors.

Carbon dioxide laser induction of heat shock protein 70 synthesis: Comparison with high temperature treatment

In a previous study, it was demonstrated that heat shock protein 70 (hsp70) was induced by short duration (<1 s) carbon dioxide (CO2) laser radiation (10.6Μm. To further characterize the stress response after laser irradiation, the time course of synthesis and cellular localization of hsp70 has been followed. As it had been shown that laser irradiation elevated the temperature to about 67±2 ‡C, the authors also attempted to duplicate the response with high temperature elevation by dipping cells grown on plastic coverslips into media heated in a hot water bath to specified temperatures. Exposure to CO2 laser irradiation resulted in a time course and localization response similar to that reported for induction of hsp70 by elevated temperature (41–44 ‡C). However, in contrast to the response to elevated temperature, only hsp70 was induced by laser irradiation. Short exposure (1–4 s) of cells medium heated to 58 ‡C produced a response similar to that obtained with CO2 laser irradiation suggesting that the CO2 laser irradiation effect on cells is produced by heating for short periods to 55–70 ‡C.

Ophthalmoscopic and pathologic description of ocular damage induced by infrared laser radiation

This study compares ocular damage effects induced by infrared (IR) lasers in the “eye-safe” wavelength range to those observed following visible wavlength laser exposures. In addition to routine fundus camera and slit-lamp observations, exposed subjects were examined via confocal scanning laser ophthalmoscopy. Histopathologic evaluation was conducted on eyes with both visible and IR laser induced focal lesions. IR laser exposure parameters which resulted in corneal, lenticular and/or retinal damage will be detailed. Unique aspects of the IR laser–tissue interaction will be discussed, including concurrent lesion formation in several tissues and secondary responses such as delayed inflammatory reactions. This latter effect may involve tissue not directly irradiated during the laser insult. Finally, implications for laser safety standards will be suggested.

Myopathic Alterations in Extraocular Muscle of Rats Subchronically Fed Pyridostigmine Bromide

To determine if alterations in extraocular muscle morphology occur after subchronic oral administration of pyridostigmine bromide, rats were continuously fed 90 mg/kg in meal and examined at 1,2, 4, 7, and 15 days. Within the first day, blood acetylcholinesterase activity was reduced by 87% and remained inhibited by 74-91% during the study. Light microscopy demonstrated that by day 1 approximately 3% of the extraocular myofibers were shrunken and invaded by inflammatory cells. The most severe degenerative changes consisting of vacuoles and inflammatory cell infiltration occurred at day 1 with progressively less severe changes at days 2 and 4. At days 7 and 15,1.3-4.5% of the myofibers still exhibited damage. Ultrastructurally, all presynaptic areas were normal but the postsynaptic areas of affected myofibers at days 1,2, and 4 showed myofilament and Z-band dissolution, mitochondrial inclusions, subneural fold and T-tubule/sarcoplasmic reticulum vacuolization and subneural fold depth reduction. By days 7 and 15, these changes were diminished in some cases and in others alterations appeared similar to day 1. We conclude that subchronic feeding of pyridostigmine bromide induces myopathic rather than neurogenic changes in rat extraocular muscle and that the myopathy is different in these muscles than in the diaphragm from the same rats.

Confocal spectral ophthalmoscopic imaging of retinal laser damage in small vertebrate eyes

We have used the small eye of the snake in combination with confocal scanning laser ophthalmoscopy to evaluate Argon laser retinal lesions. The snake eye has high optical power and high image quality. With these physiological optical attributes and confocal ophthalmic capability, we were able to examine laser induced retinal lesions over a very large dynamic range. Clear visualization of the photoreceptor matrix suggested alterations in the photoreceptor optical properties not previously noticed with conventional ophthalmoscopy and larger mammalian eyes. Inspection of anterior retinal levels suggested long term development intraretinal fibrosis and nerve fiber layer damage, as well as alteration to the choroidal vasculature at the lesion site. No alteration was observed at the retinal vascular level.

Macular hole surgery following accidental laser injury with a military rangefinder

An active duty marine corps service member had bilateral full thickness macular holes induced following accidental Q-switched laser exposure from a hand held Neodymium range finder (ANGVS-5). The right eye had a large hole nasal to the fovea, while the left eye had a much smaller hole closer to the fovea centralis. Over the 18 months following the injury, the left eye demonstrated mild progressive degradation in visual function, but retained 20/20 final visual acuity. In contrast, the hole in the right eye increased in size, developed a localized retinal detachment with cystic changes in the fovea, and had atrophy of the retinal pigment epithelium. Within 6 months after injury, acuity declined to 20/100. Macular hole surgery was performed with a goal of sealing the edges of the hole in order to allow resolution of the localized detachment and cystic changes in the fovea. In spite of surgical techniques that are generally successful in the treatment of macular holes associated with other etiologies, the fundus findings remained unchanged and visual acuity declined to 20/400. To the best of our knowledge, this is the first case report of macular hole surgery for this condition.

Confocal scanning laser ophthalmoscopic imaging resolution of secondary retinal effects induced by laser radiation

We have evaluated secondary laser induced retinal effects in non-human primates with a Rodenstock confocal scanning laser ophthalmoscope. A small eye animal model, the Garter snake, was employed to evaluate confocal numerical aperture effects in imaging laser retinal damage in small eyes vs. large eyes. Results demonstrate that the confocal image resolution in the Rhesus monkey eye is sufficient to differentiate deep retinal scar formation from retinal nerve fiber layer (NFL) damage and to estimate the depth of the NFL damage. The best comparison with histological depth was obtained for the snake retina, yielding a ratio close to 1:1 compared to 2:1 for the Rhesus. Resolution in the Garter snake allows imaging the photoreceptor matrix and therefore, evaluation of the interrelationship between the primary damage site (posterior retina), the photoreceptor matrix, and secondary sites in the anterior retina such as the NFL and the epiretinal vascular system. Alterations in both the retinal NFL and epiretinal blood flow rate were observed within several minutes post Argon laser exposure. Unique aspects of the snake eye such as high tissue transparency and inherently high contrast cellular structures, contribute to the confocal image quality. Such factors may be nearly comparable in primate eyes suggesting that depth of resolution can be improved by smaller confocal apertures and more sensitive signal processing techniques.

MORPHOLOGIC ALTERATIONS IN RAT RETINA AFTER HYPERVOLEMIC INFUSION OF CROSS-LINKED HEMOGLOBIN*

Hypervolemic infusion in rats of bis (3,5-dibromosalicyl) fumarate cross-linked hemoglobin (DBBF-Hb) to 40-60% of blood volume produced histologic lesions in retina which were not observed in rats similarly infused with human serum albumin or lactated Ringers solution. Rats treated with 40% DBBF-Hb, exhibited intermittent zones of dense retinal pigmented epithelium while 60% DBBF-Hb animals exhibited severe inner retinal edema and retinal pigmented epithelium vacuolization, large focal zones of photoreceptor outer segment disruption and in one animal, subretinal hemorrhage. Light microscopic immunocytochemical evaluation of retinas with antibodies directed to human hemoglobin and albumin, showed the presence of both hemoglobin and albumin in this tissue. Transmission electron microscopy of the lesions demonstrated vacuolated retinal pigmented epithelial cells and large areas of focal photoreceptor outer segment disruption. We conclude that hypervolemic infusion disrupts the blood retinal barrier and that although both DBBF Hb and albumin cross, only hemoglobin produced damage in the retina.

Laser induced thermal injury of rabbit cornea and treatment with anti-inflammatory agents

A moderately severe thermal injury of the central cornea of 48 Dutch-belted rabbit eyes was produced with a carbon (CO2) laser. The lesions were photographed with a slit lamp (SL) camera immediately following the injury and at 1, 2, 4, 7, 14, 21, 30 and 60 days after the exposure. Lesion size, opaqueness, and depth were graded clinically by SL biomicroscopy at the same intervals. No significant differences were found (p ⩽ 0.05) between groups of eyes treated with flurbiprofen (0.03%), prednisolone acetate (1%), and vehicle control four-times-a-day for three weeks following injury. Additionally, eyes were studied histopathologically at 3 and 60 days following injury by light and transmission electron microscopy, and clinically at 30 and 60 days by endothelial specular microscopy. Important clinical and histopathological findings included coagulative necrosis of the corneal epithelium, epithelial sloughing, fusion of stromal collagen, stromal edema and inflammatory cell infiltration, stromal scar formation, corneal thinning, endothelial hyperplasia and metaplasia, fibrinous anterior chamber reaction with hypopyon, and retrocorneal fibrous membrane formation.