Dutch-belted Rabbits Research Articles

Influence of ridge preservation procedures on extraction socket healing under antiresorptive therapy: An experimental study in rabbits.

To assess the influence of ridge preservation procedures on the healing of extraction sockets under antiresorptive therapy. A total of 10 Dutch Belted rabbits were randomly allocated to either the intravenous administration of amino-bisphosphonate (zoledronic acid) (Za) (n = 5) or a negative control group (no Za [nZa]) (n = 5). At 6 months, the mandibular and maxillary molars were extracted and the four experimental sites randomly allocated to the following subgroups: (a) socket grafting using a collagen-coated natural bone mineral (BOC) + primary wound closure, (b) coronectomy (CO), or (c) spontaneous healing + primary wound closure (SP). Za medication was continued for another 4 months. Histomorphometrical analyses considered, for example, crestal hard tissue closure of the extraction site (C) and mineralized tissue (MT) formation. Za-SP was associated with an incomplete median C (31.76% vs 100% in nZa-SP) and signs of bone arrosion along the confines of the socket. BOC had no major effects on increases in C and MT values in the Za group. CO commonly resulted in an encapsulation and partial replacement resorption of residual roots by MT without any histological signs of osteonecrosis. (a) Za-SP was commonly associated with a compromised socket healing and signs of osteonecrosis, (b) BOC had no major effect on socket healing in the Za group, and (c) CO at noninfected teeth might be a feasible measure for the prevention of a Za-related osteonecrosis of the jaw.

Efficacy of greater auricular and auriculotemporal nerve blocks performed in rabbits

ObjectiveTo evaluate the efficacy, duration and safety of greater auricular and auriculotemporal nerve blocks in rabbits. Study designProspective, randomized, crossover, experimental study. AnimalsA total of 11 healthy adult Dutch-belted rabbits. MethodsThe rabbits underwent general anesthesia and injections of the greater auricular and auriculotemporal nerves using either bupivacaine (0.3 mL kg–1, 0.5%) or the same volume of saline. After anesthesia, the efficacy and duration of nerve blocks were assessed using analgesiometry forceps on the pinna and cotton-tipped applicators within the vertical ear canal. Rabbits were monitored for abnormal carriage of the ear and auriculopalpebral nerve block of the ipsilateral eye. Body weight, food intake, fecal output and fecal pellet diameter were measured daily for 6 days after the nerve blocks were performed and compared with baseline to assess short-term effects. ResultsThe greater auricular nerve was successfully blocked in 12/16 (75%) ears for mean ± standard deviation duration of 88 ± 52 minutes. In successfully blocked ears, altered ear position was noted in five/16 (31%) cases. The auriculotemporal nerve was blocked in one/16 ears for 120 minutes. The auriculopalpebral nerve was inadvertently blocked in three/16 ears. Food intake and fecal output decreased significantly during the treatment day but returned to pretreatment values after 24 hours. There was no change in body weight or fecal pellet diameter for either treatment. Conclusions and clinical relevanceThe results suggest that duration of the greater auricular nerve block with 0.5% bupivacaine was short-lived in the live rabbit. Auriculotemporal nerve block was only achieved in one ear; therefore, further studies are warranted to evaluate the contribution of these blocks in the pain management of rabbits undergoing auricular surgery.

Transscleral cyclophotocoagulation and its histological effects on the conjunctiva

Micropulse transscleral cyclophotocoagulation (MP-TCP) is increasingly being used as an initial procedure prior to conjunctival filtration surgeries. However, it is uncertain whether MP-TCP may cause inflammation and scarring of the bulbar conjunctiva. Thus, we aimed to study the histological effects of MP-TCP (compared to controls and continuous wave [CW]-TCP) on the conjunctiva. Our study included 10 Dutch Belted Rabbits that underwent TCP in their right eyes (n = 5, MP-TCP; n = 5, CW-TCP), while their left eyes served as controls. The rabbits were euthanised at 4 weeks, and their dissected globes underwent histopathological and immunohistochemical examination. We observed greater conjunctival inflammation in MP-TCP or CW-TCP-treated eyes compared to controls, but not between each other. The majority of the lymphocytic infiltrates were CD4 T-cells. Increased conjunctival fibrosis was evident in MP-TCP or CW-TCP-treated eyes, to similar extents, compared to controls. However, the increased staining for myofibroblasts was not statistically significant in TCP-treated eyes. We concluded that MP-TCP causes significantly greater overall conjunctival inflammation and scarring compared to controls, similar to CW-TCP. As these are risk-factors for fibrosis and failure of the conjunctival bleb, further studies are required to explore the effect, if any, of post-TCP conjunctival changes on future bleb morphology and survival.

Paired measurements of cochlear function and hair cell count in Dutch-belted rabbits with noise-induced hearing loss

The effects of noise-induced hearing loss have yet to be studied for the Dutch-belted strain of rabbits, which is the only strain that has been used in studies of the central auditory system. We measured auditory brainstem responses (ABRs), 2f1−f2 distortion product otoacoustic emissions (DPOAEs), and counts of cochlear inner and outer hair cells (IHCs and OHCs, respectively) from confocal images of Myo7a-stained cochlear whole-mounts in unexposed and noise-overexposed, Dutch-belted, male and female rabbits in order to characterize cochlear function and structure under normal-hearing and hearing-loss conditions. Using an octave-band noise exposure centered at 750 Hz presented under isoflurane anesthesia, we found that a sound level of 133 dB SPL for 60 min was minimally sufficient to produce permanent ABR threshold shifts. Overexposure durations of 60 and 90 min caused median click-evoked ABR threshold shifts of 10 and 50 dB, respectively. Susceptibility to overexposure was highly variable across ears, but less variable across test frequencies within the same ear. ABR and DPOAE threshold shifts were smaller, on average, and more variable in male than female ears. Similarly, post-exposure survival of OHCs was higher, on average, and more variable in male than female ears. We paired post-exposure ABR and DPOAE threshold shift data with hair cell count data measured in the same ear at the same frequency and cochlear frequency location. ABR and DPOAE threshold shifts exhibited critical values of 46 and 18 dB, respectively, below which the majority of OHCs and IHCs survived and above which OHCs were wiped out while IHC survival was variable. Our data may be of use to researchers who wish to use Dutch-belted rabbits as a model for the effects of noise-induced hearing loss on the central auditory system.

Experimental model to evaluate the benefits of lutein to prevent retinal phototoxicity during pars plana vitrectomy surgery using xenon source light illumination in rabbits

BackgroundTo evaluate the benefits of lutein in preventing retinal phototoxicity generated by xenon light sources during vitreoretinal surgery.MethodsA prospective cross-sectional study in pigmented rabbit eyes exposed to different vitreoretinal surgery lighting simulations. Twenty Dutch-belted rabbits were divided into two groups exposed to two different xenon wavelength light sources filters (420 nm and 435 nm). In addition, two subgroups were administered with daily supplemental of 10 mg of Lutein systemically. Electroretinography (ERG), optical coherence tomography (OCT) and fluorescein angiography (FA) were performed before and after surgery to quantify the retinal damage.ResultsAll animals submitted to the experiment presented some degree of phototoxicity independent of wavelength light filter used. Retinal damage was evident as the FA presented areas of hyperfluorescence, and the OCT depicted increased reflective areas of the inner and outer retinal layers, and RPE. ERG showed a diffuse reduction of the a and b waves amplitudes in all animals.ConclusionUse of systemic administration of lutein showed no benefit to avoiding retinal phototoxicity generate to xenon light source using filters of 435 nm and 420 nm when comparing to the control group.

Topical application of povidone-iodine/dimethylsulfoxide ophthalmic gel preparation in Dutch-Belted rabbits

Purpose: To determine the ocular and systemic toxicity of a novel, topically applied ophthalmic gel preparation of povidone-iodine (PVP-I) and dimethylsulfoxide (DMSO) in Dutch-Belted rabbits.Materials and Methods: Rabbits were administered doses of the test material or control by ocular instillation four times/eye/day, 7 d/week, for a minimum of 14 consecutive days. Dosing consisted of instillation of 50 µl of the appropriate test material solution or control material (saline) into each eye of the rabbit. On the last dose of the day, 250 µl of the appropriate test material solution or control material was applied to the eyelids of each eye.Results: Treatment-related clinical signs observed during the study were limited to mild non-inflammatory changes to the eyelids and eyelashes. There was no associated pathology upon histological examination of ocular or systemic tissues. Body weights and body weight gains were unaffected by treatment. Evaluation of clinical pathology profiles (haematology, coagulation, and clinical chemistry) did not reveal any test article-related toxicity and there were no macroscopic or microscopic findings at the terminal sacrifice.Conclusions: The compositions studied in the present investigation were developed to enable repeat-dosed application to the ocular surface and periocular skin surfaces without ocular, skin or systemic toxicity. The PVP-I/DMSO compositions tested did not cause any toxicity to the ocular surface or the periocular skin. Systemic toxicity from the preparations under study was not observed in any histological or gross pathological examination.

Safety Assessment of Formulation Vehicles Following Intravitreal Administration in Rabbits.

Evaluate 21 formulation vehicles administered to rabbits after intravitreal injection for tolerability and safety. Forty-two Dutch Belted rabbits were anesthetized, and the eyes received a single intravitreal injection of the excipient formulation. Clinical signs and ocular irritation responses were recorded twice daily for 7days and microscopic evaluation of the eyes, optic nerve, and eyelids was completed at 1-week post treatment. Saline (≥ 300mOsm and ≤ 592mOsm at pH7.0 or 300mOsm at pH8.0) and 10 formulation excipients; (10% w/v PEG 3350 at pH7.4, 1% polysorbate 21 at pH7.4, PVA at pH7.0, 0.2% polysorbate 80 at pH7.2, 0.2% Pluronic F108® at pH7.3, 2%, 100mM sodium sulfate at pH3.2, 2mM sodium glycocholate at pH7.4, and 275mM D-mannitol pH7.0 in sterile water, and 100mM sodium phosphate in combination with 0.9% NaCl 300mOsm and 0.01% or 0.05% polysorbate 80 at pH7.4) considered as formulation vehicles for intravitreal injectables, were well-tolerated in rabbits. Clinical signs were transient and microscopic changes were not observed. Of the 21 formulation vehicles evaluated, 10 formulation vehicles were well-tolerated in rabbits and feasible candidates for future investigations.

In Vivo Pharmacokinetics of Bromfenac Ophthalmic Solution 0.075%, Bromfenac Ophthalmic Solution 0.07%, and Nepafenac/Amfenac Ophthalmic Suspension 0.3% in Rabbits

IntroductionLittle is known of the ocular distribution characteristics of currently branded non-steroidal anti-inflammatory drugs (NSAIDs) in the United States. This study was designed to predict the ocular bioavailability characteristics in humans using Dutch Belted rabbits as a surrogate. Commercially available, topically-applied NSAIDs containing bromfenac or nepafenac/amfenac were evaluated.Methods126 healthy adult Dutch Belted rabbits were randomly assigned to three treatment cohorts (BromSite® twice daily [BID] in the right eye, BromSite® once daily [QD] in the right eye, Prolensa® QD in the right eye and Ilevro™ QD in the left eye) and 7 post-dosing time points (0.5, 1, 2, 4, 8, 12, 24 h after final instillation). The study eyes received 40 µL of the assigned drug for a consecutive 9 days. Samples of aqueous humor, iris-ciliary body, choroid, sclera, and retina were harvested from the study eyes at the assigned time point after the last dose on the 9th day. NSAID content in ocular tissues was analyzed using high-performance liquid chromatography (HPLC), and area under the curve (AUC0.5–24h), maximum concentration (Cmax), and time to maximum concentration (Tmax) were determined.ResultsPeak NSAID concentrations were reached within 1–3 h in the anterior segment and within 1–3 h in the posterior segment after last dose. Throughout the ocular tissues, both AUC and Cmax for BromSite® (BID and QD) were consistently higher than respective NSAID concentrations of Prolensa® QD and Ilevro® QD. When comparing BromSite® BID to QD, the BID regimen produced generally higher but statistically similar bromfenac concentrations throughout the ocular tissues except in the aqueous humor and iris-ciliary body, where the AUC BID was statistically significantly higher with BromSite® BID.ConclusionAs a surrogate to human ocular bioavailability, BromSite® demonstrated significantly greater NSAID compared to Prolensa® QD and Ilevro® QD. The DuraSite® component of BromSite® appears to enhance ocular penetration throughout both anterior and posterior tissues.FundingSun Pharmaceutical Industries Ltd.

Ocular Toxicity Profile of ST-162 and ST-168 as Novel Bifunctional MEK/PI3K Inhibitors.

ST-162 and ST-168 are small-molecule bifunctional inhibitors of MEK and PI3K signaling pathways that are being developed as novel antitumor agents. Previous small-molecule and biologic MEK inhibitors demonstrated ocular toxicity events that were dose limiting in clinical studies. We evaluated in vitro and in vivo ocular toxicity profiles of ST-162 and ST-168. Photoreceptor cell line 661W and adult retinal pigment epithelium cell line ARPE-19 were treated with increasing concentrations of bifunctional inhibitors. Western blots, cell viability, and caspase activity assays were performed to evaluate MEK and PI3K inhibition and dose-dependent in vitro toxicity, and compared with monotherapy. In vivo toxicity profile was assessed by intravitreal injection of ST-162 and ST-168 in Dutch-Belted rabbits, followed by ocular examination and histological analysis of enucleated eyes. Retinal cell lines treated with ST-162 or ST-168 exhibited dose-dependent inhibition of MEK and PI3K signaling. Compared with inhibition by monotherapies and their combinations, bifunctional inhibitors demonstrated reduced cell death and caspase activity. In vivo, both bifunctional inhibitors exhibited a more favorable toxicity profile when compared with MEK inhibitor PD0325901. Novel MEK and PI3K bifunctional inhibitors ST-162 and ST-168 demonstrate favorable in vitro and in vivo ocular toxicity profiles, supporting their further development as potential therapeutic agents targeting multiple aggressive tumors.

The use of micro-CT imaging to examine and illustrate fetal skeletal abnormalities in Dutch Belted rabbits and to prove concordance with Alizarin Red stained skeletal examination.

In our laboratory we evaluated the use of micro-computed tomography (micro-CT) using a high resolution acquisition protocol and fetuses obtained on Gestation Day (GD) 29 (mating = GD 0). To show concordance between traditional Alizarin Red S stain and micro-CT skeletal examination methods, 103 fetuses from 19 untreated Dutch belted rabbits were obtained by cesarean section and stored frozen. The fetuses were thawed, imaged and examined digitally by micro-CT, then stained and re-examined using traditional methods. A total of 12 individual malformations and 35 unique variations were detected by both methods. Differences in the extent of ossification were found in only 51 of 26,196 bones while 99.8% of the observations were identical. Of the 51 differences, 31 were an unossified fifth medial phalanx of the forepaw indicating that very low-density skeletal bones may be visible by Alizarin Red stain but not by micro-CT scan. To establish this methodology under pharmaceutical testing conditions, we obtained and imaged by micro-CT Alizarin Red S stained abnormal fetal rabbit skeletons previously exposed to a drug candidate associated with craniofacial malformations in humans. All of the types of skeletal abnormalities first identified by staining were also detected by micro-CT examination. Representative images of these 66 different fetal skeletal abnormalities were characterized, and compiled to illustrate visual concordance between micro-CT scanned and traditional Alizarin Red S stained skeletons. Micro-CT imaging is an accurate, reliable and robust method that can be used as an alternative to stain when examining fetal rabbit skeletons in developmental toxicity studies.

Sealing of Corneal Lacerations Using Photoactivated Rose Bengal Dye and Amniotic Membrane.

Watertight closure of perforating corneoscleral lacerations is necessary to prevent epithelial ingrowth, infection, and potential loss of the eye. Complex lacerations can be difficult to treat, and repair with sutures alone is often inadequate. In this study, we evaluated a potentially sutureless technology for sealing complex corneal and scleral lacerations that bonds the amniotic membrane (AM) to the wound using only green light and rose bengal dye. The AM was impregnated with rose bengal and then sealed over lacerations using green light to bond the AM to the deepithelialized corneal surface. This process was compared with suture repair of 3 laceration configurations in New Zealand White rabbits in 3 arms of the study. A fourth study arm assessed the side effect profile including viability of cells in the iris, damage to the blood-retinal barrier, retinal photoreceptors, retinal pigment epithelium, and choriocapillaris in Dutch Belted rabbits. Analyses of the first 3 arms revealed a clinically insignificant increase in polymorphonuclear inflammation. In the fourth arm, iris cells appeared unaffected and no evidence of breakdown of the blood-retinal barrier was detected. The retina from green light laser-treated eyes showed normal retinal pigment epithelium, intact outer segments, and normal outer nuclear layer thickness. The results of these studies established that a light-activated method to cross-link AM to the cornea can be used for sealing complex penetrating wounds in the cornea and sclera with minimal inflammation or secondary effects.

Experimental verification of subthreshold laser therapy using conventional pattern scan laser.

PurposeLeading-edge therapeutic laser technologies are not available at every medical facility; therefore, alternative approaches incorporating novel advances in digital and laser technology into more readily available conventional methods have generated significant research interest. Using a rabbit model, this study investigated whether the algorithm used in the Endpoint Management (EM) software system of the latest devices could enable subthreshold laser treatment in conventional retinal tissue laser therapy systems.Materials and methodsTwo types of devices were used, the PASCAL Streamline 577 and the MC 500-Vixi™, and the laser method was classified into three categories: EM; single-shot using PASCAL with arbitrary energy settings (PSS-SDM); and MC500-VixiTM (VX-SDM), which were performed in eight eyes from four Dutch-Belted rabbits. In EM, 100 mW (100%) was set as a landmark, and the laser energy parameters were gradually decreased to 80%, 60%, 50%, 40%, 30%, 20%, and 10%, using a 2 × 3 square pattern. In PSS-SDM and VX-SDM, as control, the laser energy was gradually decreased to 100, 80, 60, 50, 40, 30, 20, and 10 mW. The laser settings were fixed at 200 μm, 20 ms, and a wavelength of 577 μm. To identify and compare the extent of tissue damage at each spot size, optical coherence tomography (OCT) and histological findings were used to construct a three-dimensional histopathology image using a confocal laser scanning fluorescence microscope.ResultsThe spot size at 50% setting on EM was 7183 μm2; PSS-SDM required 50 mW (5503 μm2) to 60 mW (10279 μm2) and VX-SDM required 50 mW (7423 μm2) to create the approximate spot size. Furthermore, at 50 mW of PSS-SDM and VX-SDM, the extent of tissue damage in all three methods was generally in accord with the outer nuclear layer by OCT and inner nuclear layer by histopathological imaging.ConclusionThese findings suggest that it may be possible to perform subthreshold laser therapy using approximations from the EM algorithm.

Consequences of Puberty on Efficacy of Intraocular Pressure-Lowering Drugs in Male Dutch-Belted Rabbits.

To investigate the changes in intraocular pressure (IOP), aqueous flow, and outflow facility, as well as efficacy of IOP-lowering drugs before and after sexual development in rabbits. Male Dutch-belted rabbits were studied at night between the ages of 8 and 44 weeks. During these times, body weight, testicular volume, and serum testosterone were measured to monitor sexual maturity. Ocular measurements included anterior chamber depth, central corneal thickness, IOP, aqueous flow, and outflow facility. Systemic acetazolamide or topical timolol, latanoprost, or saline were administered pre- and postpuberty to assess drug effects on these parameters. Body weight, testicular volume, and serum testosterone increased until 28 weeks of age. IOP increased during prepuberty (R2 = 0.49, P = 0.003), dropped significantly during puberty, rising again immediate postpuberty, and changing little thereafter. Postpuberty compared with prepuberty found higher IOP (P < 0.0001), slower aqueous flow (P = 0.008), lower outflow facility (not statistically significant, P = 0.07), increased central cornea thickness, and increased anterior chamber volume. Timolol lowered IOP both pre- and postpuberty, whereas, latanoprost and acetazolamide decreased IOP postpuberty only. As male rabbits mature, the cornea thickens and the anterior chamber volume increases. At the same time, aqueous flow slows, yet, IOP increases. This suggests that decreased outflow facility and/or increased episcleral venous pressure might contribute to the puberty-related changes in IOP. Underdevelopment of tissues of the outflow pathways may contribute to the differences in drug efficacy in rabbits when young compared with after sexual maturity.

Axonal Conduction Delays, Brain State, and Corticogeniculate Communication.

Thalamocortical conduction times are short, but layer 6 corticothalamic axons display an enormous range of conduction times, some exceeding 40-50 ms. Here, we investigate (1) how axonal conduction times of corticogeniculate (CG) neurons are related to the visual information conveyed to the thalamus, and (2) how alert versus nonalert awake brain states affect visual processing across the spectrum of CG conduction times. In awake female Dutch-Belted rabbits, we found 58% of CG neurons to be visually responsive, and 42% to be unresponsive. All responsive CG neurons had simple, orientation-selective receptive fields, and generated sustained responses to stationary stimuli. CG axonal conduction times were strongly related to modulated firing rates (F1 values) generated by drifting grating stimuli, and their associated interspike interval distributions, suggesting a continuum of visual responsiveness spanning the spectrum of axonal conduction times. CG conduction times were also significantly related to visual response latency, contrast sensitivity (C-50 values), directional selectivity, and optimal stimulus velocity. Increasing alertness did not cause visually unresponsive CG neurons to become responsive and did not change the response linearity (F1/F0 ratios) of visually responsive CG neurons. However, for visually responsive CG neurons, increased alertness nearly doubled the modulated response amplitude to optimal visual stimulation (F1 values), significantly shortened response latency, and dramatically increased response reliability. These effects of alertness were uniform across the broad spectrum of CG axonal conduction times.SIGNIFICANCE STATEMENT Corticothalamic neurons of layer 6 send a dense feedback projection to thalamic nuclei that provide input to sensory neocortex. While sensory information reaches the cortex after brief thalamocortical axonal delays, corticothalamic axons can exhibit conduction delays of <2 ms to 40-50 ms. Here, in the corticogeniculate visual system of awake rabbits, we investigate the functional significance of this axonal diversity, and the effects of shifting alert/nonalert brain states on corticogeniculate processing. We show that axonal conduction times are strongly related to multiple visual response properties, suggesting a continuum of visual responsiveness spanning the spectrum of corticogeniculate axonal conduction times. We also show that transitions between awake brain states powerfully affect corticogeniculate processing, in some ways more strongly than in layer 4.

Determination of a No-Observable Effect Level for Endotoxin Following a Single Intravitreal Administration to Dutch Belted Rabbits.

The purpose of this study was to characterize the inflammatory response and determine a no-observable effect level (NOEL) in rabbit eyes after endotoxin intravitreal (ITV) injection. Fifty-three naïve male Dutch Belted rabbits were treated with a single 50-μL ITV injection ranging from 0.01 to 0.75 endotoxin units/eye (EU/eye) and monitored for up to 42 days post treatment. Ophthalmic examination included slit-lamp biomicroscopy and indirect ophthalmoscopy. Laser flare photometry was performed in a subset of animals. On days 2, 8, 16, and 43, a subset of animals was necropsied and eyes processed for histopathological evaluation. Intravitreal injection of endotoxin at ≥0.05 EU/eye resulted in a dose-related anterior segment inflammation response. No aqueous flare or cell response was noted in the 0.01 EU/eye dose group. A more delayed posterior segment response characterized by vitreal cell response was observed beginning on day 5, peaking on day 9, and decreasing starting on day 16 that persisted at trace to a level of 1+ on day 43. Microscopy findings of infiltrates of minimal mixed inflammatory cells in the vitreous and subconjunctiva and proteinaceous fluid in the anterior chamber and/or vitreous were observed in eyes given ≥0.1 EU/eye. We defined the NOEL for ITV endotoxin to be 0.01 EU/eye, suggesting that the vitreal cavity is more sensitive to the effects of endotoxin than the anterior segment and aqueous chamber. These data highlight the importance of assessing endotoxin level in intravitreal formulations, as levels as low as 0.05 EU/eye may confound the safety evaluations of intravitreal therapeutics in rabbits.

Preclinical Acute Ocular Safety Study of Combined Intravitreal Carboplatin and Etoposide Phosphate for Retinoblastoma.

To describe the ocular toxicity of intravitreal carboplatin and etoposide phosphate (VP16P) in Dutch-Belted rabbits. Twenty-two adult male Dutch-Belted rabbits (Cohort 1) each received a single, bilateral intravitreal injection (0.05 mL). For Cohort 1, safety was assessed via electroretinograms (ERGs) and ocular examination. Of nine total groups in Cohort 1, the first five received the following single agents: Group 1: normal saline; Group 2: VP16P 75 µg; Group 3: VP16P 100 µg; Group 4: carboplatin 4 µg; and Group 5: carboplatin 8 µg. Groups 6 through 9 received the following combination of carboplatin/ VP16P, respectively: Group 6: 8 µg/75 µg, Group 7: 8 µg/50 µg, Group 8: 4 µg/50 µg, and Group 9: 2 µg/25 µg. Cohort 2 consisted of 15 Dutch-Belted rabbits in seven groups (Groups 10 through 16), each receiving a single, bilateral intravitreal injection. For Cohort 2, safety was assessed via histopathology. Groups 2 through 8 demonstrated a statistically significant decrease (relative to Group 1) in at least one ERG waveform amplitude obtained 4 weeks postinjection (P < .05). Group 9 (carbo 2 µg/VP16P 25 µg) did not manifest ERG toxicity. Fundoscopic toxicity consisted of slight-to-moderate attenuation of vessels in rabbits receiving doses above carbo 4 µg/VP16P 50 µg. Histopathologic retinal toxicity (Cohort 2) was dose-dependent, ranging from full-thickness atrophy in rabbits receiving the highest dose to normal in rabbits receiving carbo 2 µg/VP16P 25 µg. Combined carboplatin and VP16P may be compatible for intravitreal injection therapy, and a single dose of 2 µg/25 µg appears to be safe in a rabbit model. These agents may be a safer alternative to intravitreal melphalan (Alkeran; GlaxoSmithKline, Brentford, United Kingdom) for the treatment of vitreous seeds in retinoblastoma. [Ophthalmic Surg Lasers Imaging Retina. 2017;48:151-159.].

The Feasibility of Spectral-Domain Optical Coherence Tomography Grading of Anterior Chamber Inflammation in a Rabbit Model of Anterior Uveitis.

PurposeTo determine the feasibility and accuracy of spectral-domain optical coherence tomography (SD-OCT) based grading of anterior chamber cell, using aqueous sampling as a standard, in a rabbit model of anterior uveitis.MethodsAdult Dutch-belted rabbits were preimmunized with M. tuberculosis (Tb) H37RA antigen, 1 week prior to induction of anterior uveitis with an intracameral injection of Tb antigen. The anterior chamber was imaged with SD-OCT, followed by a slit lamp examination. Two independent, trained graders recorded their estimate of anterior chamber cell count using the Standardization of Uveitis Nomenclature (SUN) scores for each eye prior to performing an anterior chamber tap to determine the aqueous cell density using a hemocytometer. Using the aqueous cell density as a standard, correlation with SD-OCT counts were compared to those with SUN scores.ResultsOverall, SD-OCT correlated well with the hemocytometer counts (Spearman coefficient = 0.53, P < 0.001) compared with SUN grading and hemocytometer counts (Spearman coefficient = 0.02, P = 0.88). The correlation improved to 0.65 (P < 0.001) when we excluded eyes with corneal thickness ≥ 470 μm. Eyes with corneal thickness ≥ 470 μm exhibited the greatest degree of ocular inflammation and corneal opacity.ConclusionsIn our rabbit model, SD-OCT grading of anterior chamber cell correlated significantly better with aqueous cell counts, compared to traditional slit lamp grading. Spectral-domain optical coherence tomography grading of anterior chamber cell may be a good alternative to SUN grading. Although SUN grading remains the clinical gold standard, alternative quantitative methods to assess ocular inflammation could provide insight into disease mechanism and aid in measuring treatment response.

Morphologic changes in the retina after selective retina therapy.

To investigate structural changes in the retina by histologic evaluation and in vivo spectral domain optical coherence tomography (SD-OCT) following selective retina therapy (SRT) controlled by optical feedback techniques (OFT). SRT was applied to 12 eyes of Dutch Belted rabbits. Retinal changes were assessed based on fundus photography, fluorescein angiography (FAG), SD-OCT, light microscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM) at each of the following time points: 1h, and 1, 3, 7, 14 and 28days after SRT. BrdU (5'-bromo-2'-deoxy-uridine) incorporation assay was also conducted to evaluate potential proliferation of RPE cells. SRT lesions at1 h after SRT were ophthalmoscopically invisible. FAG showed leakage in areas corresponding to SRT lesions, and hyperfluorescence disappeared after 7days. SD-OCT showed that decreased reflectivity corresponding to RPE damage was restored to normal over time in SRT lesions. Histologic analysis revealed that the damage in SRT lesions was primarily limited to the retinal pigment epithelium (RPE) and the outer segments of the photoreceptors. SEM and TEM showed RPE cell migration by day 3after SRT, and restoration of the RPE monolayer with microvilli by 1week after SRT. At 14 and 28days, ultrastructures of the RPE, including the microvilli and tight junctions, were completely restored. The outer segments of the photoreceptors also recovered without sequelae. Interdigitation between the RPE and photoreceptors was observed. BrdU incorporation assay revealed proliferation of RPE on day 3after SRT, and peak proliferation was observed on day 7after SRT. Based on multimodal imaging and histologic assessment, our findings demonstrate that SRT with OFT could selectively target the RPE without damaging the neurosensory retina. Therefore, the use of SRT with OFT opens the door to the possibility of clinical trials of well-defined invisible and nondestructive retina therapy, especially for macular disease.

Discovery of the ROCK inhibitor netarsudil for the treatment of open-angle glaucoma

Inhibition of Rho kinase (ROCK) to improve fluid outflow through the trabecular meshwork and lower intraocular pressure is a strategy for the development of new anti-glaucoma agents. Alpha-aryl-beta-amino isoquinoline analogs were identified as potent ROCK inhibitors. Compounds that provided a longer duration of intraocular pressure reduction in Dutch Belted rabbits also inhibited norepinephrine transporter. Ester 60 improved bioavailability of its parent ROCK inhibitor, 29 (Ki=0.2nM) and demonstrated an effective and sustained IOP reduction for 24h after dosing. From these studies, netarsudil (a.k.a. AR-13324) was discovered and is currently in clinical trials for the treatment of glaucoma and ocular hypertension.

A Probability Analysis of Historical Pregnancy and Fetal Data from Dutch Belted and New Zealand White Rabbit Strains from Embryo-Fetal Development Studies.

Embryo-fetal development (EFD) studies, typically in pregnant rats and rabbits, are conducted prior to enrolling females of reproductive age in clinical trials. Common rabbit strains used are the New Zealand White (NZW) and Dutch Belted (DB). As fetal abnormalities can occur in all groups, including controls, Historical Control Data (HCD) is compiled using data from control groups of EFD studies, and is used along with each study's concurrent control group to help determine whether fetal abnormalities are caused by the test article or are part of background incidences. A probability analysis was conducted on 2014 HCD collected at Charles River Inc., Horsham PA on Covance NZW, Covance DB, and Charles River (CR) NZW rabbits. The analysis was designed to determine the probability of 2 or 3 out of a group of 22 does aborting their litter or of having a fetal abnormality by chance. Results demonstrate that pregnancy parameters and fetal observations differ not only between strains, but between sources of rabbits of the same strain. As a result the probability of these observations occurring by chance in two or three litters was drastically different. Although no one single strain is perfect, this analysis highlights the need to appreciate the inherent differences in pregnancy and fetal abnormalities between strains, and points out that an apparent isolated increased incidence of an observation in one strain will not necessarily be test-article related in another strain. A robust HCD is critical for interpretation of EFD rabbit studies, regardless of the rabbit strain used.