Ocular and systemic toxicity of intravitreal topotecan in rabbits for potential treatment of retinoblastoma.

Treatment of intraocular retinoblastoma with vitreous seeding is a challenge. Different routes of chemotherapy administration have been explored in order to attaining pharmacological concentrations into the posterior chamber. Intravitreal drug injection is a promissing route for maximum bioavailability to the vitreous but it requires a well defined dose for achieving tumor control while limited toxicity to the retina. Topotecan proved to be a promising agent for retinoblastoma treatment due to its pharmacological activity and limited toxicity. High and prolonged concentrations were achieved in the rabbit vitreous after 5 µg of intravitreal topotecan. However, whether a lower dose could achieve potentially therapeutic levels remained to be determined. Thus, we here study the pharmacokinetics of topotecan after 0.5 µg and the toxicity profile of intravitreal topotecan in the rabbit eye as a potential treatment of retinoblastoma. A cohort of rabbits was used to study topotecan disposition in the vitreous after a single dose of 0.5 µg of intravitreal topotecan. In addition, an independent cohort of non-tumor bearing rabbits was employed to evaluate the clinical and retinal toxicity after four weekly injections of two different doses of intravitreal topotecan (Group A, 5 µg/dose; Group B, 0.5 µg/dose) to the right eye of each animal. The same volume (0.1 ml) of normal saline was administered to the left eye as control. A third group of rabbits (Group C) served as double control (both eyes injected with normal saline). Animals were weekly evaluated for clinical and hematologic values and ocular evaluations were performed with an inverse ophthalmoscope to establish potential topotecan toxicity. Weekly controls included topotecan quantitation in plasma of all rabbits. Electroretinograms (ERGs) were recorded before and after topotecan doses. One week after the last injection, topotecan concentrations were measured in vitreous of all eyes and samples for retinal histology were obtained. Our results indicate that topotecan shows non linear pharmacokinetics after a single intravitreal dose in the range of 0.5-5 µg in the rabbit. Vitreous concentration of lactone topotecan was close to the concentration assumed to be therapeutically active after 5 h of 0.5 µg intravitreal administration. Eyes injected with four weekly doses of topotecan (0.5 or 5 µg/dose) showed no significant differences in their ERG wave amplitudes and implicit times in comparison with control (p > 0.05). Animals showed no weight, hair loss or significant changes in hematologic values during the study period. There were no significant histologic damage of the retinas exposed to topotecan treatments. After intravitreal administration no topotecan could be detected in plasma during the follow-up period nor in the vitreous of treated and control animals after 1 week of the last injection. The present data shows that four weekly intravitreal injection of 5 µg of topotecan is safe for the rabbit eye. Despite multiple injections of 0.5 µg of topotecan are also safe to the rabbit eye, lactone topotecan vitreous concentrations were potentially active only after 5 h of the administration. We postulate promising translation to clinics for retinoblastoma treatment.

Eyelid lymphoma in a patient with a gold weight implant. / Linfoma palpebral en paciente con implante de pesa de oro.

CASE REPORT: A male patient with an exposure keratopathy caused by lagophthalmos. A gold weight was implanted in the right upper eyelid. Eight months later, he presented with erythema and swelling of right upper eyelid. An incisional biopsy was performed, reporting extranodal marginal zone B cell lymphoma. DISCUSSION: when a tumour at the site of a gold weight implant is refractory to treatment, it is essential to perform an incisional biopsy to establish the histopathological diagnosis. Ocular adnexal lymphomas are relatively common. The presence of foreign material can cause chronic inflammation that could be the stimulus for the development of a lymphoproliferative disorder.

On the Cornea of Healthy Merino Sheep: A Detailed Ex Vivo Confocal, Histological and Ultrastructural Study.

Our study performed qualitative and quantitative studies on the corneal ultrastructure of healthy female Merino sheep of ages 4 months and 6 years old from the Argentinean Pampa. The corneas were evaluated using ex vivo laser-scanning confocal microscopy, light microscopy and transmission electron microscopy. Those studies allowed us to obtain detailed images of the corneal layers as well as quantitative data of the cellular and sub-basal nerve densities in the cornea from sheep of different ages. The density of the corneal cells was significantly different in the anterior versus the posterior epithelium and stroma. Moreover, the density of the epithelial, stromal cells and endothelial cells, as well as the sub-basal nerve density were significantly lower in adult than in young animals. Our work provided a wide-ranging description of the corneal ultrastructure of healthy female Merino sheep, which adds to the current knowledge about the ophthalmological aspects of this species and undoubtedly benefits veterinarians.

Intracellular inclusions in meningothelial meningioma. A histochemical and ultrastructural study.

This is a study on the histochemical and electron microscope findings of the intracellular inclusions in a case of meningothelial meningioma of the frontoethmoidal region with orbital involvement. The inclusions are PAS-positive, diastase resistant, and stain strongly positive with the modified Morel-Sisley reaction for protein-bound tyrosine and weakly with dihydroxy-dinaphthyl-disulfide (DDD) for the demonstration of sulfhydryl (SH) and disulfide (S-S) groups of proteins. These staining reactions suggest the inclusions are composed of conjugated proteins (glycoproteins). Ultrastructurally, the inclusions are located within intracellular spaces lined by microvilli and are composed of granular material, usually forming a dense core that is intermixed with small vacuoles. Numerous desmosomes and whorls of tonofibrils are concentrated around them. Our findings support the view that the intracellular inclusions, which are rarely found in meningiomas, represent an active secretory product of the meningothelial cells rather than a degenerative or phagocytic process.

Hemangiopericytoma of the orbit: a clinicopathologic study of 30 cases.

The clinical and pathologic features of 30 orbital hemangiopericytomas were reviewed. An effort was made to determine the clincopathologic characteristics that may correlate with the biologic behavior. Histopathologic criteria evaluated included degree of cellularity, nuclear atypia, mitotic activity, tumor size, and the presence of hemorrhage and necrosis. Histopathologically, 16 tumors were classified as benign, five as borderline, and nine as malignant. Follow-up information was obtained in 27 cases (mean duration of follow-up period was five and one-half years). Eight tumors recurred (30 per cent). Five patients died, four with widespread metastases (15 per cent) and one of unrelated causes. Tumors that recurred or metastasized were distributed among all three histologic groups. The interval between the onset of symptoms and metastasis for three of the four lethal tumors was 31 years, 28 years, and 8 1/2 years; in the fourth case it was unknown. For the entire series, the five-year actuarial survival rate was 89 per cent. Although the number of cases is too small to serve as a basis for firm conclusions, the lack of unequivocal correlation between the histologic features and clinical behavior is consistent with the unpredictable behavior of this neoplasm, as concluded by other investigators. Because of the apparent circumscription of most of these tumors, complete surgical excision rather than exenteration is recommended. Metastasis usually develops late in the clinical course of the disease, indicating that long-term follow-up studies are required before a cure can be assumed.

Benign fibrous tumor of the choroid.

A 32-year-old white woman presented with an amelanotic choroidal tumor in the left eye. Although a malignant melanoma could not be totally ruled out, some features suggested a benign tumor. The patient was followed up without treatment during a 2-year period. Neovascular glaucoma developed and the blind eye was enucleated. Histopathologic examination revealed a benign fibrous tumor of the choroid, presumably a fibrous histiocytoma.

Sympathetic ophthalmia after pars plana vitrectomy-lensectomy for endogenous bacterial endophthalmitis.

A 71-year-old woman developed an endogenous bacterial endophthalmitis in her right eye. Because of the rapid progression she was treated by para plana vitrectomy-lensectomy and with antibiotics and corticosteroids. The patient improved after the antibiotic therapy. One month later she had a recurrence of the inflammatory signs in the same eye. Examination disclosed lens remnants in the vitreous. Because of the possibility of phacoallergic uveitis, she underwent a second vitrectomy. The corticosteroid dosage was increased. She did well until three months later when, while still taking corticosteroids, she noted loss of visual acuity in the left eye, accompanied by bilateral inflammatory signs. Sympathetic ophthalmia was suspected and the blind right eye was enucleated. The choroid was grossly unremarkable; the routine sections disclosed many inconspicuous foci of mononuclear infiltration. Tangential sections of flat-embedded choroid, however, showed ill-defined nests of epithelioid cells containing phagocytosed melanin, supporting the diagnosis of sympathetic ophthalmia. To our knowledge, this is the first published report of this complication after pars plana vitrectomy-lensectomy in the absence of nonsurgical trauma or other surgical procedures.

Sjogren's syndrome: a comparative study of impression cytology of the conjunctiva and buccal mucosa, and salivary gland biopsy.

The diagnosis of Sjogren's syndrome (SJ) as an underlying disease of keratoconjunctivitis sicca is important because of the many ocular and systemic complications. We compared the results of impression cytology of conjunctiva (ICC) and the results of labial salivary gland biopsy (LSB) with impression cytology of buccal mucosa (ICB) in 33 patients with SJ. LSB, ICC, and ICB were considered positive in all patients. Moderate to severe changes were graded in 85% of the biopsy specimens, 94% of the conjunctival specimens, and 76% of the buccal mucosa specimens. The rate of agreement between LSB and ICB was 97%. The use of ICB in the clinical ophthalmological examination may be helpful in patients with consistent history, and clinical and ocular findings, before the biopsy procedure.

Atypical corneal dystrophy with stromal amyloid deposits.

Corneal dystrophies are distinctive clinically and histopathologically; however, variations do occur. We present two cases of stromal amyloidosis from one family masquerading other corneal dystrophy. The two cases are from a six-generation family with an autosomal dominant corneal dystrophy resembling Reis-Bucklers' dystrophy. In these cases, neither the propositus nor other family members showed typical lattice lines. Light and electron microscopy of the obtained corneal buttons disclosed amyloid stromal deposits. The clinicopathologic correlation of these cases suggests that this family represents a variant of stromal amyloid dystrophies.

In vivo lamellar keratoplasty using platelet-rich plasma as a bioadhesive.

PURPOSE: The use of standard sutures has been replaced by platelet-rich plasma (PRP), a bioadhesive agent, in several surgical procedures. This prompted us to test PRP efficacy in experimental lamellar keratoplasty. METHODS: After lamellar anterior keratoplasty, PRP with a mean concentration of 807,564 platelets/mm(3) was used to attach the corneal flap to the stromal surface in 12 New Zealand white rabbits. 10-0 nylon sutures were used in one control group of 12 animals and no suture was used in a second control group of six rabbits. Animals were killed at days 2, 7, 30, and 90 for histological and smooth muscle actin (SMA) immunohistochemical analysis. RESULTS: The PRP group showed a tight corneal graft from the first postoperative hours until they were killed. A transparent cornea was seen at 30 days and remained clear until the end of the protocol. Histological specimens showed no signs of ocular inflammation in any animal within the PRP group. Electron microscopy showed normal morphological features on the flap and stromal bed, and a clear interface zone without cells or debris. The number of stromal myofibroblasts was lower than that seen in the suture group at 3 months postsurgery. The sutured group showed an attached cornea with signs of inflammation around the knots. All flaps without PRP or sutures were completely detached in the immediate postoperative period. CONCLUSIONS: PRP was useful for attaching the corneal flap and it was well tolerated by the rabbit corneal tissue. Corneal healing was satisfactory. Further studies on PRP adhesiveness in grafts with donor corneas should be performed before considering its use in patients.

Participación de componentes inmunológicos en la etiopatogenia de la queratopatía climática esferoidea / Involvement of immune components in the etiopathogenia of climatic droplet keratopathy

Objetivo. Investigar si componentes de la inmunidad innata están involucrados en la iniciación/perpetuación de las anormalidades estructurales observadas en la capa de Bowman y el estroma superficial de la córnea de pacientes con queratopatía climática esferoidea (QCE). Materiales y métodos. En el estudio participaron 8 pacientes con QCE y 12 individuos sanos del Departamento El Cuy, Provincia de Río Negro, y 10 individuos sanos de la ciudad de Córdoba. Todos ellos, luego de firmar el consentimiento informado, recibieron un examen oftalmológico completo y se recolectaron muestras de lágrima para estudiar las concentraciones de diferentes citocinas, niveles y formas de metaloproteinasas de matriz (MMPs), y el inhibidor natural de MMPs (TIMP-1). Se realizó microscopía confocal in vivo (MCF) en algunos pacientes y controles. Biopsias de córneas provenientes de pacientes que fueron tratados con queratoplastia penetrante también fueron estudiadas mediante inmunohistoquímica (IHQ). Resultados. Los resultados de MCF indicaron claramente una progresión en la cantidad de depósitos a nivel subepitelial, a medida que la enfermedad avanza. El daño progresivo de las fibras nerviosas sub basales y estromales en los estadios 2 y 3 se correlaciona con pérdida de la sensibilidad corneal. Además de estas alteraciones, observamos que el número de células dendríticas (CD) en el limbo corneal aumentó significativamente a medida que la QCE progresa. En lágrimas de pacientes con QCE se detectaron concentraciones significativamente superiores de citocinas proinflamatorias (IL1ß e IL-8) que en individuos controles (p<0,005). No se halló IL-2, IL-17, IL-4, IL-13 ni IL-10 en pacientes y ni controles. Las actividades de gelatinasas (MMP-9 y -2) fueron significativamente mayores en QCE que en los controles (p<0,001), mientras que los niveles de TIMP-1 fueron significativamente menores en los pacientes (p<0,05). La concentración de MMP-8 fue mayor en controles pero los niveles de esta colagenasa-2 fueron 30 veces superiores, tanto en QCE como controles, con respecto a los valores de los individuos de un centro urbano. Mediante IHC observamos reactividad para MMP-9 en la mayoría de las células epiteliales, solamente en córneas con QCE. Conclusión. Demostramos un rol protagónico del eje citocinas proinflamatorias - gela-tinasas en el desarrollo de la QCE. Los altos niveles de IL-1ß e IL-8 en lágrimas de pacientes facilitan la producción de MMP-8 y gelatinasas, y los efectos de las mismas se exacerban, ya que los pacientes tienen bajos niveles de sus inhibidores naturales (TIMP-1). La MMP-9, además de degradar componentes de la matriz extracelular, cataliza la activación postranscripcional de IL-1ß, potenciando el proceso inflamatorio. Estos resultados son los primeros en explicar mecanismos inmunológicos involucrados en la etiopatogénesis de la QCE y aportan nuevas alternativas para el desarrollo de terapias preventivas utilizando inhibidores de IL-1ß y/o gelatinasas(AU)

Objective. To investigate whether components of innate immunity are involved in the initiation / perpetuation of the structural abnormalities observed in Bowman's layer and superficial stroma of the córnea of patients with Climatic droplet keratopathy (CDK). Materials and Methods. The study included 8 CDK patients and 12 healthy individuals from Department El Cuy, Province of Río Negro, and 10 healthy subjects from the city of Córdoba. All of them, after signing informed consent, received a thorough eye exam and tear samples were collected to study the concentrations of different cytokines, and levels and forms of matrix metalloproteinases (MMPs) and their natural inhibitor (TIMP-1). In vivo confocal microscopy (CFM) was performed in some patients and controls. Corneal biopsies from CDK patients treated with penetrating keratoplasty were also studied by immunohistochemistry (IHC). Results. CFM results clearly indicated a progression in the amount of deposits at corneal sub epithelial level as the disease progresses. The progressive damage in the nerve plexus in stages 2 and 3 correlated with a loss of corneal sensitivity. In addition to these alterations, we observed that the number of dendritic cells (DC) in the limbus increased significantly as the disease progresses.In tears of patients with CDK we detected significantly higher concentrations of pro-inflammatory cytokines (IL-1ß and IL-8) than in control subjects (p < 0.005). We found no IL-2, IL-17, IL-4, IL-13 and IL-10 in patients and controls. The activities of gelatinases (MMP-9 and -2) were significantly higher in CDK than in controls (p < 0.001), while TIMP-1 levels were significantly lower in patients (p < 0.05). The concentration of MMP-8 was higher in controls, but levels of this collagenase-2 were 30 times higher, both in CDK and controls, with respect to MMP-8 values of individuals inhabiting an urban area. By IHC we observed reactivity for MMP-9 in most epithelial cells only in CDK corneas. Conclussion. We demonstrated a key role of the axis pro-inflammatory cytokines ­ gelatinases in the development of CDK. High levels of IL-1ß and IL-8 in tears of patients facilitate the production of MMP-8 and gelatinases, and the effects of these molecules are exacerbated because patients have low levels of their natural inhibitors (TIMP-1). Since MMP-9 besides degrading extracellular matrix components, catalyzes the post translational activation of IL-1ß, the inflammatory process is fuelled. These results are the first to explain immunological mechanisms involved in the pathogenesis of the QCE and provide new alternatives for the development of preventive therapies using inhibitors of IL-1ß and / or gelatinases.(AU)

Unusual ciliary body tumor. Mesectodermal leiomyoma.

A 23-year-old white woman presented with a pigmented ciliary body mass in the left eye. The tumor appeared to transilluminate when observed with indirect illumination. Clinically the tumor was considered to represent either a malignant melanoma or a cyst and was excised by iridocyclectomy. Electron microscopic examination established the smooth muscle nature of the tumor. The literature on this unusual tumor is reviewed. Furthermore, this report serves to emphasize the value of iridocyclectomy in the diagnosis and treatment of ciliary body tumors.

Cavitary melanocytoma of the ciliary body.

The clinical and pathologic findings of a patient with an unusual cavitary melanocytoma of the ciliary body are presented. Clinically and with ultrasonography the lesion appeared to have a cystic component. Differential diagnoses included a malignant melanoma or a cyst, and the lesion was excised by iridocyclectomy.