Ellison Bentley

Nanoscale Topography–Induced Modulation of Fundamental Cell Behaviors of Rabbit Corneal Keratocytes, Fibroblasts, and Myofibroblasts

PURPOSE Keratocyte-to-myofibroblast differentiation is a key factor in corneal wound healing. The purpose of this study was to determine the influence of environmental nanoscale topography on keratocyte, fibroblast, and myofibroblast cell behavior. METHODS Primary rabbit corneal keratocytes, fibroblasts, and myofibroblasts were seeded onto planar polyurethane surfaces with six patterned areas, composed of anisotropically ordered grooves and ridges with a 400-, 800-, 1200-, 1600-, 2000-, and 4000-nm pitch (pitch = groove + ridge width). After 24 hours cells were fixed, stained, imaged, and analyzed for cell shape and orientation. For migration studies, cells on each patterned surface were imaged every 10 minutes for 12 hours, and individual cell trajectories and migration rates were calculated. RESULTS Keratocytes, fibroblasts, and myofibroblasts aligned and elongated to pitch sizes larger than 1000 nm. A lower limit to the topographic feature sizes that the cells responded to was identified for all three phenotypes, with a transition zone around the 800- to 1200-nm pitch size. Fibroblasts and myofibroblasts migrated parallel to surface ridges larger than 1000 nm but lacked directional guidance on submicron and nanoscale topographic features and on planar surfaces. Keratocytes remained essentially immobile. CONCLUSIONS Corneal stromal cells elongated, aligned, and migrated, differentially guided by substratum topographic features. All cell types failed to respond to topographic features approximating the dimensions of individual stromal fibers. These findings contribute to our understanding of corneal stromal cell biology in health and disease and their interaction with biomaterials and their native extracellular matrix.

Electron Microscopy of the Canine Corneal Basement Membranes

The purpose of this study was to characterize the surface topographical features of the epithelial and endothelial (Descemet’s) basement membranes of the canine cornea. Corneas were obtained from young, healthy dogs (<2 years old) with no history or evidence of previous ocular disease. The epithelium and endothelium was carefully removed preserving the anterior and posterior basement membranes. The specimens were examined by transmission electron microscopy and scanning electron microscopy. The epithelial and endothelial basement membrane surface topography is an intricate meshwork of pores and fibers measuring in the nanometer size range. The features of the endothelial basement membrane overall are smaller in size than the epithelial basement membrane. These surface topographical features may incite changes in epithelial and endothelial cell behavior.

Effects of the Application of Neck Pressure by a Collar or Harness on Intraocular Pressure in Dogs

The effect on intraocular pressure (IOP) from dogs pulling against a collar or a harness was evaluated in 51 eyes of 26 dogs. The force each dog generated while pulling against a collar or a harness was measured. Intraocular pressure measurements were obtained during application of corresponding pressures via collars or harnesses. Intraocular pressure increased significantly from baseline when pressure was applied via a collar but not via a harness. Based on the results of the study, dogs with weak or thin corneas, glaucoma, or conditions for which an increase in IOP could be harmful should wear a harness instead of a collar, especially during exercise or activity.

The pharmacologic assessment of a novel lymphocyte function-associated antigen-1 antagonist (SAR 1118) for the treatment of keratoconjunctivitis sicca in dogs.

PURPOSE Keratoconjunctivitis sicca (KCS) is characterized by inflammation and decreased production of tears containing increased levels of cytokines. The release occurs in the setting of conjunctival and lacrimal gland inflammation, potentially mediated by the interaction between lymphocyte function-associated antigen (LFA)-1, a cell surface protein found on lymphocytes, and its cognate ligand intercellular adhesion molecule (ICAM)-1. SAR 1118 is a novel LFA-1 antagonist and may be an effective therapeutic agent for the treatment of KCS. The following studies were performed to assess the in vitro activity of SAR 1118 and to evaluate the clinical efficacy of topical SAR 1118 for the treatment of idiopathic canine KCS. METHOD Pharmacodynamics were assessed by measuring the ability of SAR 1118 to inhibit Jurkat T-cell binding with recombinant human ICAM-1 and to inhibit cytokine release from human peripheral blood mononuclear cells (PBMCs) stimulated by staphylococcal enterotoxin B. For the assessment of clinical efficacy, 10 dogs diagnosed with idiopathic KCS were treated with SAR 1118 1% topical ophthalmic solution three times daily for 12 weeks. Schirmers tear test (STT) was used to measure tear production. RESULTS SAR 1118 demonstrated concentration-dependent inhibition of Jurkat T-cell attachment, inhibition of lymphocyte activation, and release of inflammatory cytokines, particularly the Th1, Th2, and Th17 T-cell cytokines IFN-γ, IL-2, and IL-17F, respectively. Mean STT values increased from 3.4 mm during week 1 to 5.8 mm at week 12 (P < 0.025). No SAR 1118-related adverse events were observed. CONCLUSIONS SAR 1118 appears to be an effective anti-inflammatory treatment for KCS. Additional studies are warranted to establish the efficacy of SAR 1118 for the treatment of KCS in humans.

Effectiveness of injection of local anesthetic into the retrobulbar space for postoperative analgesia following eye enucleation in dogs

OBJECTIVE To assess the efficacy of a retrobulbar bupivacaine nerve block for postoperative analgesia following eye enucleation in dogs. DESIGN Randomized controlled trial. ANIMALS 22 dogs. PROCEDURES Client-owned dogs admitted to the hospital for routine eye enucleation were enrolled with owner consent and randomly assigned to a treatment (bupivacaine hydrochloride) or control (saline [0.9% NaCl] solution) group. Baseline subjective pain scores were recorded. Anesthesia consisted of hydromorphone and midazolam preoperatively, thiopental or propofol for induction, and isoflurane in oxygen for maintenance. An inferior-temporal palpebral retrobulbar injection of either saline solution or bupivacaine was administered. Transpalpebral eye enucleation was performed. Pain scores were recorded at 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after extubation (time 0) by observers masked to treatment groups. Dogs were given hydromorphone (0.2 mg/kg [0.09 mg/lb], IM or IV) as a rescue analgesic if the subjective pain score totaled >or= 9 (out of a maximum total score of 18) or >or= 3 in any 1 category. RESULTS 9 of 11 control dogs required a rescue dose of hydromorphone, but only 2 of 11 dogs in the bupivacaine treatment group required rescue analgesia. Mean time to treatment failure (ie, administration of rescue analgesia following extubation) was 0.56 hours (95% confidence interval, 0.029 to 1.095 hours) for the 11 dogs that received hydromorphone. CONCLUSIONS AND CLINICAL RELEVANCE Retrobulbar administration of bupivacaine in dogs in conjunction with traditional premedication prior to eye enucleation was an effective form of adjunctive analgesia and reduced the need for additional postoperative analgesics.

Refractive states of eyes and association between ametropia and breed in dogs

OBJECTIVE To assess the refractive state of eyes in various breeds of dogs to identify breeds susceptible to ametropias. ANIMALS 1,440 dogs representing 90 breeds. PROCEDURES In each dog, 1 drop of 1% cyclopentolate or 1% tropicamide was applied to each eye, and a Canine Eye Registration Foundation examination was performed. Approximately 30 minutes after drops were administered, the refractive state of each eye was assessed via streak retinoscopy. Dogs were considered ametropic (myopic or hyperopic) when the mean refractive state (the resting focus of the eye at rest relative to visual infinity) exceeded +/- 0.5 diopter (D). Anisometropia was diagnosed when the refractive error of each eye in a pair differed by > 1 D. RESULTS Mean +/- SD refractive state of all eyes examined was -0.05 +/- 1.36 D (emmetropia). Breeds in which the mean refractive state was myopic (< or = -0.5 D) included Rottweiler, Collie, Miniature Schnauzer, and Toy Poodle. Degree of myopia increased with increasing age across all breeds. Breeds in which the mean refractive state was hyperopic (> or = +0.5 D) included Australian Shepherd, Alaskan Malamute, and Bouvier des Flandres. Astigmatism was detected in 1% (14/1,440) of adult (> or = 1 year of age) dogs; prevalence of astigmatism among German Shepherd Dogs was 3.3% (3/90). Anisometropia was detected in 6% (87/1,440) of all dogs and in 8.9% (8/90) of German Shepherd Dogs. CONCLUSIONS AND CLINICAL RELEVANCE Refractive states of canine eyes varied widely and were influenced by breed and age. In dogs expected to have high visual function (eg, performance dogs), determination of refractive state is recommended prior to intensive training.

Evaluation of topical nalbuphine or oral tramadol as analgesics for corneal pain in dogs: a pilot study

OBJECTIVE To evaluate the effectiveness of topical nalbuphine or oral tramadol in the treatment of corneal pain in dogs. ANIMALS STUDIED Fourteen male Beagle dogs. PROCEDURES Dogs were divided into three treatment groups and sedated with dexmedetomidine (5 μ/kg IV). A 4 mm corneal epithelial wound was created in the right eye (OD) of all dogs. Sedation was reversed with atipamazole IM. All dogs received pre/post ophthalmic examinations. Post operatively, Group NB (n = 5) received topical 1% preservative-free nalbuphine OD q8 h and an oral placebo PO q8 h. Group TR (n = 5) received tramadol (4 mg/kg) PO q8 h and topical sterile saline OD q8 h. Group CNTRL (n = 4) received topical sterile saline OD q8 h and an oral placebo q8 h. All dogs received topical 0.3% gentamicin OD TID until healed. Dogs were pain scored using a pain scoring system modified from the University of Melbourne pain scale at 0, 1, 2, 4, and 6 h, then every 6 h by observers masked to treatment, until corneal wounds were healed. Treatment failure was recorded if cumulative pain scores were above a minimum threshold of acceptable pain and rescue analgesia of morphine (1.0 mg/kg IM) was administered subsequently. RESULT Four dogs in Group NB, one dog in Group TR, and two dogs in Group CNTRL required rescue analgesia. There was no significant difference in the incidence of treatment failure between groups (P = 0.184). Mean time to rescue was 9.16 h. All corneal wounds were healed by 84 h. CONCLUSIONS The results of this study suggest tramadol rather than nalbuphine should be further investigated for the treatment of corneal pain.

Ocular lesions associated with systemic hypertension in dogs: 65 cases (2005–2007)

OBJECTIVE To characterize ocular findings in hypertensive dogs, determine prevalence of hypertension in dogs with ocular disease suggestive of hypertension, and examine possible relationships between degree of hypertension and ocular disease. DESIGN Retrospective case series. ANIMALS 65 dogs initially referred for blood pressure measurement (n = 22), ophthalmic examination (25), or both (18). PROCEDURES Medical records were reviewed to identify dogs examined at the teaching hospital that underwent a complete ophthalmic examination and blood pressure measurement within a 24-hour period between January 1, 2005, and December 31, 2007. Signalment, history, blood pressure measurements, ophthalmic examination findings, and any vasoactive drug treatments were recorded. Ocular lesions considered likely to be associated with systemic hypertension included retinal hemorrhage, retinal detachment, hyphema, tortuous vessels, and subretinal edema. RESULTS Of the 65 dogs, 42 were hypertensive (systolic blood pressure ≥ 160 mm Hg) and 23 were normotensive. Sixty-two percent (26/42) of hypertensive dogs had ≥ 1 type of ocular lesion identified. Retinal hemorrhage was the most common ocular lesion in hypertensive dogs (17/42 [40%]). The presence of ≥ 1 type of ocular lesion had moderate sensitivity and specificity of 62% and 61 %, respectively, for identification of hypertension. Fifteen of the 25 (60%) dogs referred for blood pressure measurement after initial ophthalmic examination were found to be hypertensive. CONCLUSIONS AND CLINICAL RELEVANCE Ocular lesions are common in dogs with systemic hypertension. Dogs with hypertension or diseases associated with hypertension should be monitored carefully for evidence of ocular target organ damage, and hypertension should be systematically ruled out in dogs with characteristic ocular lesions.

Expression of matrix metalloproteinase 2 and 9 in experimentally wounded canine corneas and spontaneous chronic corneal epithelial defects

Purpose: To determine matrix metalloproteinase (MMP) 2 and MMP 9 expression in acute and chronic experimentally wounded canine corneas and keratectomy samples from canine patients with spontaneous chronic corneal epithelial defects (SCCEDs). Methods: Mechanical debridement was performed unilaterally in 25 healthy dogs for the acute wound study. Twenty-four hours (n = 8), 48 hours (n = 5), 72 hours (n = 3), or 1 week (n = 9) after wounding, the dogs were euthanized. Debridement was performed once weekly for 8 weeks for the chronic study (n = 8). Therapeutic superficial keratectomies (n = 16) were performed on SCCED patients. Gelatin zymography and immunohistochemistry were performed. Results: Acute wounds showed upregulation of MMP 9 at all time points. At 7 days after wounding, values diminished markedly but remained elevated above those of unwounded controls. SCCED and chronic wound samples showed a significant increase in MMP 9 compared with controls but were less than that of acute wounds. There was no significant difference between chronic wounds versus SCCED samples. Fellow control eyes showed significant upregulation of MMP 9 in tandem with wounded eyes. There was no significant difference in values for MMP 2 in wounded corneas or SCCED compared with those of controls. Immunhistochemistry localized MMP 9 to predominantly the epithelium with some staining of keratinocytes and stroma. Conclusions: The dog exhibits similar MMP expression during corneal wound healing to that of other species. The lack of significant difference in MMP expression between SCCED and chronic wounds suggest that MMP 2 and 9 are not involved in the pathophysiology of SCCED and are more likely altered secondary to a chronic epithelial defect.

Biosynthetic Corneal Substitute Implantation in Dogs

Purpose: To assess integration of a biosynthetic corneal implant in dogs. Methods: Three normal adult laboratory Beagles underwent ophthalmic examinations, including slit-lamp biomicroscopy, indirect ophthalmoscopy, applanation tonometry, and Cochet-Bonnet aesthesiometry. Biosynthetic corneas fabricated from glutaraldehyde crosslinked collagen and copolymers of collagen and poly(N-isopropylacrylamide-co-acrylic acid-co-acryloxysuccinimide, denoted as TERP) were implanted into dogs by a modified epikeratoplasty technique. Ophthalmic examinations and aesthesiometry were performed daily for 5 days and then weekly thereafter for 16 weeks. Corneal samples underwent histopathological and transmission electron microscopy examination at 16 weeks. Results: Implants were epithelialized by 7 days. Intraocular pressure was within normal range throughout the study. Aesthesiometry values dropped from an average of 3.67 cm preoperatively to less than 1 mm for all dogs for the first postoperative weeks. By week 16, the average Cochet-Bonnet value was 1.67 cm, demonstrating partial recovery of functional innervation of the implant. No inflammation or rejection of the implant occurred, and minimal haze formation was noted. Light microscopy revealed thickened but normal epithelium over the implant with fibroblast migration into the scaffold. On transmission electron microscopy, the basement membrane was irregular but present and adhesion complexes were noted. Conclusion: Biosynthetic corneal implantation is well tolerated in dogs, and the collagen-polymer hybrid construct holds promise for clinical application in animals and humans.