Performance Assessment of New Urinary Translational Safety Biomarkers of Drug-induced Renal Tubular Injury in Tenofovir-treated Cynomolgus Monkeys and Beagle Dogs.

Newer urinary protein kidney safety biomarkers can outperform the conventional kidney functional biomarkers blood urea nitrogen (BUN) and serum creatinine (SCr) in rats. However, there is far less experience with the relative performance of these biomarkers in dogs and nonhuman primates. Here, we report urine protein biomarker performance in tenofovir-treated cynomolgus monkeys and beagle dogs. Tenofovir intravenous daily dosing in monkeys for 2 or 4 weeks at 30 mg/kg/day resulted in minimal to moderate tubular degeneration and regeneration, and tenofovir disoproxil fumarate oral dosing in dogs for 10 days at 45 mg/kg/day resulted in mild to marked tubular degeneration, necrosis, and regeneration. Among biomarkers tested, kidney injury molecule 1 (Kim-1) and clusterin (CLU) clearly outperformed BUN and SCr and were the most reliable in detecting the onset and progression of tenofovir-induced tubular injury. Cystatin C, retinol binding protein 4, ß2-microglobulin, neutrophil gelatinase-associated lipocalin, albumin, and total protein also performed better than BUN and SCr and added value when considered together with Kim-1 and CLU. These findings demonstrate the promising utility of these urinary safety biomarkers in monkeys and dogs and support their further evaluation in human to improve early detection of renal tubular injury.

Long Term Temporal Changes in Structure and Function of Rat Visual System After Blast Exposure.

Purpose: We identify long-term ocular sequelae subsequent to experimental blast exposure. Methods: Male Long-Evans rats were exposed to 230 kPa side-on primary blast overpressure using a compressed air driven shock tube. Visual system function and structure were assessed for 8 weeks after exposure using optokinetic nystagmus and optical coherence tomography. Vitreous protein expression and histology (TUNEL, H&E) were performed at 1 day and 1, 4, and 8 weeks. IOP was recorded bilaterally during blast in a subset of animals. Results: Blast pressure profiles resembled the Friedlander waveform indicative of an open field blast. Peak IOP in directly-exposed eyes (240 kPa) was similar to peak blast overpressure, but IOP in indirectly-exposed eyes was 30% lower. Contrast sensitivity of blast-exposed animals decreased significantly by 20% 1 day after blast and did not recover by 8 weeks. Significant swelling and structural damage to the corneal epithelial and stromal layers were observed 2 weeks after blast exposure. Swollen corneas increased 254 ± 143 µm from baseline by 6 weeks, and scarring developed by 8 weeks. Histology revealed additional lens pathology 1 week after blast, suggestive of cataract development. Endothelial cell density increased significantly in blast-exposed animals between 1 and 4 weeks. Neurofilament heavy chain significantly increased after blast and returned to near baseline values by 8 weeks. Inflammatory cytokine changes corroborated ocular pathology findings. Conclusions: These data demonstrate immediate visual dysfunction and biochemical responses, but delayed structural pathology, in response to single primary blast exposure. The delayed pathology time course may provide a window to implement treatment strategies for improved visual outcome.

Cyclic Head Rotations Produce Modest Brain Injury in Infant Piglets.

Repetitive back-and-forth head rotation from vigorous shaking is purported to be a central mechanism responsible for diffuse white matter injury, subdural hemorrhage, and retinal hemorrhage in some cases of abusive head trauma (AHT) in young children. Although animal studies have identified mechanisms of traumatic brain injury (TBI) associated with single rapid head acceleration-decelerations at levels experienced in a motor vehicle crash, few experimental studies have investigated TBI from repetitive head rotations. The objective of this study was to systematically investigate the post-injury pathological time-course after cyclic, low-velocity head rotations in the piglet and compare them with single head rotations. Injury metrics were the occurrence and extent of axonal injury (AI), extra-axial hemorrhage (EAH), red cell neuronal/axonal change (RCNAC), and ocular injury (OI). Hyperflexion/extension of the neck were purposefully avoided in the study, resulting in unscaled angular accelerations at the lower end of reported infant surrogate shaking kinematics. All findings were at the mild end of the injury spectrum, with no significant findings at 6 h post-injury. Cyclic head rotations, however, produced modest AI that significantly increased with time post-injury (p < 0.035) and had significantly greater amounts of RCNAC and EAH than noncyclic head rotations after 24 h post-injury (p < 0.05). No OI was observed. Future studies should investigate the contributions of additional physiological and mechanical features associated with AHT (e.g., hyperflexion/extension, increased intracranial pressure from crying or thoracic compression, and more than two cyclic episodes) to enhance our understanding of the causality between proposed mechanistic factors and AHT in infants.

Molecular umbrella conjugate for the ocular delivery of siRNA.

The synthesis, computer modeling, and biological activity of an octawalled molecular umbrella short interfacing RNA (siRNA) conjugate is described. This molecular umbrella-siRNA conjugate exhibited mRNA knockdown activity in vitro in the absence of a transfection reagent. Evaluation of this molecular umbrella conjugate in vivo, using the rat eye via intravitreal injection, resulted in sequence specific mRNA knockdown in the retina with no obvious signs of toxicity, as judged by ophthalmic examination.

Intravitreal cidofovir injection for the management of chronic glaucoma in dogs.

OBJECTIVE: The objective of this retrospective study was to determine whether an intravitreal injection of 562.5 ug of cidofovir was effective in lowering the intraocular pressure (IOP) in chronically glaucomatous avisual canine eyes. PATIENTS: This study reviews the medical records of 167 canine patients diagnosed with chronic glaucoma between 2006 and 2011. The criteria for study inclusion were patients with irreversibly avisual eyes that were refractory to medical management, without evidence of endophthalmitis or intraocular neoplasia as determined by historical, clinical, and/or ultrasonographic examination, who were treated with an intravitreal injection of cidofovir. PROCEDURE: Using only topical anesthesia for most patients, aqueouscentesis was followed by an intravitreal injection of 562.5 ug of cidofovir and 1 mg of subconjunctival triamcinolone. An initial recheck was performed 14 days following the injection. RESULTS: The mean IOP immediately prior to injection was 46 ± 13 mm Hg with a median of 47 mm Hg. At the initial recheck, the mean IOP was 10 ± 11 mm Hg with a median of 6 mm Hg. A common postinjection finding was mild intraocular inflammation, which was controlled in all cases with topical neopolydex solution. Post injection complications included corneal ulceration (2.4%) and endophthalmitis (2.4%). Phthisis bulbi was a common sequalae (70%). One injection of cidofovir was successful in lowering the IOP in 85% of cases (P < 0.0001); multiple injections raised the success rate to 97%. CONCLUSIONS: An intravitreal injection of cidofovir was found to be an effective method of lowering intraocular pressure to <20 mm Hg. While the common side effect of phthisis bulbi resulted in a less than optimal cosmetic result, patients subjectively benefited from enhanced comfort warranting consideration of this approach in the management of chronically glaucomatous avisual canine eyes.

Use of bovine pericardium (Tutopatch®) graft for surgical repair of deep melting corneal ulcers in dogs and corneal sequestra in cats.

OBJECTIVE: To evaluate the efficacy of bovine pericardium (BP) graft in the treatment of deep melting corneal ulcers in three dogs and corneal sequestra in three cats. PROCEDURE: Three dogs with keratomalacia affecting the deep third of the stroma and three cats with corneal sequestrum were evaluated and underwent surgery. Following keratectomy, BP material was placed into the keratectomy bed and sutured to the recipient cornea with 9/0 polyglactin suture material. Postoperative treatment with topical and systemic antibiotics, systemic nonsteroidal anti-inflammatory agents, and topical atropine was prescribed. Follow-up examinations were carried out 1, 2 weeks, 1 and 2 months after the surgery and consisted of a complete ophthalmic examination. Optical coherence tomography (OCT) was performed 1 and 2 months after the surgery in one dog and in one cat. RESULTS: At 1 week, corneal neovascularization was present around the BP graft in all cases. Four weeks after the BP graft, in two dogs and in all cats, the vascularization was regressing and the graft was integrated into the cornea, which was regaining transparency. Topical treatment with anti-inflammatory agents was then prescribed for 2 weeks. Two months after the surgery, 5 of 6 corneas in two dogs and three cats had healed with focal corneal scarring. The remaining dog had progression of the keratomalacia involving the deep BP graft that required additional surgery, but became blind. CONCLUSION: Bovine pericardium graft offers a promising option for surgical reconstruction of the cornea following keratectomy for the management of corneal ulcers and sequestra.

Intracranial meningioma causing partial amaurosis in a cat.

OBJECTIVE: To describe a case of intracranial meningioma causing visual impairment in a cat, successfully treated by surgery. PROCEDURES: An adult neutered male domestic cat was referred with a 10-month history of progressive visual impairment and altered behavior. Investigations included physical, ophthalmologic and neurological examinations as well as hematology, serum biochemistry and CT scan of the head. RESULTS: The menace response was absent in the left eye and decreased in the right eye. Electroretinograms were normal on both eyes, as was ophthalmic examination, ruling out an ocular cause and allowing a presumptive diagnosis of partial amaurosis due to a post-retinal lesion. CT scan demonstrated a large sessile extra axial mass along the right parietal bone and thickening of the adjacent bone. Cerebrospinal fluid was not collected because high intracranial pressure represented a risk for brain herniation. A right rostrotentorial craniectomy was performed to remove the tumor. Ten days after surgery, vision was improved, neurological examination was normal and normal behavior was restored. Ten months after surgery, ophthalmological examination showed no visual deficit and CT scan did not reveal any sign of recurrence. CONCLUSION: Advanced imaging techniques allow veterinarians to detect early cerebral diseases and to provide specific treatment when it is possible. In cases of feline amaurosis due to intracranial meningioma, the vital prognosis is good while the visual prognosis is more uncertain, but recovery of normal vision and normal behavior is possible as demonstrated in the present case.

Ocular hemorrhages in neonatal porcine eyes from single, rapid rotational events.

PURPOSE: To characterize ocular hemorrhages from single, rapid head rotations in the neonatal pig. METHODS: Three- to 5-day-old anesthetized piglets (n=51) underwent a single, rapid (117-266 rad/s) head rotation in the sagittal (n=13), coronal (n=7), or axial (n=31) planes. Six hours after injury, the animals were euthanatized and perfusion fixed, and the brain and eyes were harvested for gross and histopathologic examination by masked neuro- and ocular pathologists. RESULTS: Ocular hemorrhage was found in 73% of animals (51% bilateral). Intraocular hemorrhage was primarily located near the vitreous base (70% of injured animals had ciliary body hemorrhage, and 11% had peripheral retinal hemorrhage). Hemorrhages were also found in the anterior chamber (11%), vitreous (5%), and optic nerve (disc, 8%; nerve sheath, 57%). Rapid axial head rotations resulted in a higher incidence of intraocular hemorrhage than coronal or sagittal head rotations, but the difference did not reach statistical significance (P=0.06). Control eyes had no injuries. CONCLUSIONS: Optic nerve sheath and ciliary body hemorrhages were common in piglets that experienced a single, rapid head rotation. Retinal hemorrhage was present in a smaller number of animals. Most intraocular hemorrhages were located in regions of strong vitreous attachment, suggesting that this animal model will be useful in investigating the effect of vitreoretinal adhesion on ocular hemorrhage caused by inertial head rotations. Extrapolation of this model to the human infant should not be made until the effect of anatomic differences between the human and pig on the occurrence and patterns of ocular injuries is further investigated.

Congenital orbital cysts of neural tissue in two dogs.

A 3-month-old English Cocker Spaniel and a 6-month-old miniature poodle presented with clinical signs related to an abnormal right eye since birth. In both dogs, the right globe could not be identified and was replaced by a fluctuant intraorbital mass covered by a vascularized mucous membrane. Ultrasonography demonstrated that both masses were cystic structures markedly larger in size than the normal contralateral globes. In both cases, surgical excision revealed a multilobular cyst filling the whole orbital cavity. Histopathologic examination and immunostaining for glial fibrillary acid protein and S100 protein supported a diagnosis of neural cysts associated with ocular dysplasia. The definitive diagnosis was congenital cystic eye and microphthalmos with cyst for the Cocker Spaniel and miniature poodle, respectively. Karyotype was normal in both dogs. Congenital cystic eye and microphthalmos with cyst result from defects in early embryonic life that arise following formation of the optic vesicle and prior to closure of the optic fissure. To the authors' knowledge neither has been reported in the canine species. They should be considered in the differential diagnosis of orbital cysts in dogs.

Goniodysgenesis associated with primary glaucoma in an adult European Short-haired cat.

A 9.5-year-old, male castrated European Short-haired (ESH) cat was presented with bilateral glaucoma associated with pectinate ligament dysplasia and an open iridocorneal angle (ICA) upon gonioscopy. The right eye (OD) was avisual and slightly enlarged; the left eye (OS) was still visual. Intraocular pressure (IOP) had been controlled with medical therapy over a 1.5 year-period in both eyes (OU). Eventually IOP could not be adequately controlled medically and the painful and blind right eye was enucleated and transscleral diode laser cyclophotocoagulation was performed twice in the left eye with less than optimal results and progressive loss of vision. Histopathology of the right eye showed goniodysgenesis characterized by failure of differentiation of the pectinate ligament, which existed as a solid sheet of uveal tissue at the entrance of a hypoplastic ciliary cleft, which contained loose mucoid mesenchymal tissue. The trabecular meshwork was hypoplastic and the scleral venous plexus could not be identified. Other findings of chronic glaucoma were inner retinal atrophy, optic nerve atrophy with disc cupping, scleral thinning, peripheral corneal vascularization and pigmentation, and mild focal iridal mononuclear inflammatory cell infiltrate.

Classification of feline intraocular neoplasms based on morphology, histochemical staining, and immunohistochemical labeling.

The objectives of this study were to evaluate morphologic, histochemical, and immunohistochemical characteristics of well-differentiated and anaplastic intraocular neoplasms of cats, and to develop a diagnostic algorithm for, and investigate the association of ruptured lenses with these neoplasms. Seventy-five feline globes with intraocular neoplasms were stained with hematoxylin and eosin and examined by light microscopy. Morphologic diagnoses included 33 intraocular sarcomas, 17 diffuse iris melanomas, 15 lymphosarcomas, three ciliary adenomas, one metastatic carcinoma, and six undifferentiated intraocular neoplasms. Sections of these globes were then stained with periodic acid Schiff (PAS), and immunohistochemical (IHC) labels for various cellular markers. Histochemical staining and IHC labeling confirmed cellular differentiation in 73/75 neoplasms but was discordant with morphologic diagnoses in 8/75. These included four neoplasms morphologically diagnosed as lymphosarcomas but which expressed differentiation antigens consistent with melanoma (n = 3) or ciliary adenocarcinoma (n = 1), and four tumors morphologically diagnosed as intraocular sarcomas that expressed differentiation antigens for melanoma (n = 2), metastatic carcinoma (n = 1), or remained undifferentiated (n = 1). Immunohistochemical labeling suggested a diagnosis in 5/6 morphologically undifferentiated neoplasms including one intraocular sarcoma, two diffuse iridal melanomas, and two ciliary adenocarcinomas. Based upon morphologic, histochemical, and IHC characterization, ruptured lens capsules were detected in 28/30 intraocular sarcomas, 3/24 diffuse iris melanomas and 1/11 lymphosarcomas, but not in ciliary epithelial neoplasms, metastatic carcinomas, or undifferentiated intraocular neoplasms. An algorithm is provided that facilitates stain and IHC label selection for differentiating anaplastic intraocular feline neoplasms.

Repeated spike exposure to the insecticide chlorpyrifos interferes with the recovery of visual sensitivity in rats.

Reports from Japan and India and data submissions to the US EPA indicate that exposure to cholinesterase (ChE)-inhibiting organophosphorous insecticides (OP) can produce ocular toxicity, in particular long-lasting changes in retinal physiology and anatomy. We have examined the effects of a 1 year dietary exposure to the OP chlorpyrifos (CPF) on retinal structure and function. Adult male Long-Evans rats were fed CPF in their diet at the rate of 0, 1 (low), or 5 (high) mg/kg body weight/day. In addition, half of each feeding group received an oral (spike) dose of CPF in corn oil (45 mg/kg) or corn oil (VEH) alone every 2 months, resulting in six exposure groups: Control-VEH, Control-CPF, Low-VEH, Low-CPF, High-VEH, and High-CPF. Dark-adapted electroretinograms (ERG) were measured 3-5 months (n= 15-18/group) after the completion of dosing. There were no significant differences between dose or spike groups in a-wave, b-wave, or oscillatory potential amplitudes or implicit times. In addition, the time course of dark adaptation were measured in a subset of these rats (6-8/group) eight months after the completion of dosing by determining the flash intensity needed to elicit a 40 microV b-wave at selected intervals after bleaching 90% of the photopigment. Rats receiving the episodic oral spike of CPF showed a slowed recovery of dark-adapted sensitivity compared to rats receiving the corn oil VEH across chronic dosing conditions. No effects were seen on retinal morphology. This result suggests that episodic high dose exposures to CPF may result in altered retinal function. This effect, akin to effects seen in aging humans and humans exposed to other ChE-inhibiting compounds, may reflect alterations in the photoreceptors and retinal pigment epithelium (RPE) complex necessary for regenerating photopigment.

Cataract formation in a strain of rats selected for high oxidative stress.

The primary purpose of this study was to define the clinical and morphological features of cataractogenesis in the OXYS strain of rats that generate excess reactive oxygen species. Rats were sequentially examined from birth to the development of mature cataracts with slit lamp biomicroscopy. Morphology of selected stages of cataract development was studied using light and transmission electron microscopy (TEM), immunohistochemical localization of the lipid peroxidation product 4-hydroxynonenal (HNE) and fluorescent antibody labeling for DNA oxidation products. Lenses from age-matched normal rats were used as controls. OXYS rats developed cataracts as young as two weeks of age with progression to maturity by 1 year. Clinically, cataracts appeared initially either as nuclear or sub-capsular cortical changes and progressed to pronounced nuclear cataracts within months. TEM confirmed the light microscopic impression of region-specific alterations in both fiber cell cytoplasmic protein matrix and membrane structure. The outer adult nuclear region showed extensive cellular damage similar to osmotic cataracts, which is consistent with the postulated high uptake of glucose in the OXYS strain. The adult and outer fetal nuclear cells displayed several types of focal damage. The inner fetal and embryonic nuclear cells demonstrated textured cytoplasm, suggesting protein degradation or redistribution. Staining for HNE was increased in epithelium, cortex and nucleus compared to control lenses. Fluorescent antibody probes demonstrated increased levels of DNA oxidation products in OXYS rat lenses compared to age-matched controls. Fourier analysis of nuclear cytoplasm revealed significant components with corresponding sizes greater than 100 nm and, using a new theoretical approach, the texturing of the cytoplasm was shown to be sufficient to cause opacification of the nucleus. The OXYS rat appears to be an ideal model for oxidative stress cataractogenesis. The potential oxidative damage observed is extensive and characteristic of the developmental region. The source of oxidative damage may in part be a response to elevated levels of glucose. Because oxidative stress is thought to be a major factor in cataract formation in both diabetic and non-diabetic aging humans, this animal model may be a useful tool in assessing efficacy of antioxidant treatments that may slow or prevent cataract formation.

Ocular histopathologic observations in Norwegian Elkhounds with primary open-angle, closed-cleft glaucoma.

A retrospective histopathologic study of primary glaucoma in the Norwegian Elkhound was undertaken with the study of 9 clinically normal eyes and 22 glaucomatous eyes. All glaucomatous eyes showed goniodysgenesis as manifested by pectinate ligament dysplasia and/or trabecular meshwork dysplasia. Cystic degeneration of the iridociliary epithelial and/or peripheral retina was present in a high percentage of both normotensive and glaucomatous eyes. Utilizing the scheme proposed by Smith et al. (Veterinary and Comparative Ophthalmology 1993; 3: 16-28) the morphology of this disease in the Norwegian Elkhound would be classified as an open-angle, closed-cleft glaucoma, with histopathologic alterations of the outflow pathway similar to that described in other breeds with primary glaucoma.

Bilateral neuroepithelial choristomas of the optic disc in a cynomolgus monkey (Macaca fascicularis): a case report.

A 6-year-old intact male cynomolgus monkey of Chinese origin was received at the Sierra Biomedical Facility. While physical examination revealed good body condition with no abnormalities, routine ophthalmic examination revealed bilateral proliferative optic neuropathy involving the dorsal aspect of the optic disc. No changes were noted in the appearance of the lesions over 8 months, and fluoroescein angiography revealed no abnormalities other than obstruction of the view of the retinal vessels by the lesions. Histopathologic studies revealed characteristics consistent with a diagnosis of bilateral neuroepithelial choristoma.