Shin Ae Park

Periocular and Intra-Articular Injection of Canine Adipose-Derived Mesenchymal Stem Cells: An In Vivo Imaging and Migration Study

PURPOSEnImmune-mediated diseases affect millions of people worldwide with an economic impact measured in the billions of dollars. Mesenchymal stem cells (MSCs) are being investigated in the treatment of certain immune mediated diseases, but their application in the treatment of the majority of these disorders remains largely unexplored. Keratoconjunctivitis sicca can occur as a result of progressive immune-mediated destruction of lacrimal tissue in dogs and humans, and immune-mediated joint disease is common to both species. In dogs, allogeneic MSC engraftment and migration have yet to be investigated in vivo in the context of repeated injections.nnnMETHODSnWith these aims in mind, the engraftment of allogeneic canine MSCs after an injection into the periocular and intra-articular regions was followed in vivo using magnetic resonance and fluorescent imaging.nnnRESULTSnThe cells were shown to be resident near the site of the injection for a minimum of 2 weeks. Analysis of 61 tissues demonstrated preferential migration and subsequent engraftment of MSCs in the thymus as well as the gastrointestinal tract. These results also detail a novel in vivo imaging technique and demonstrate the differential spatial distribution of MSCs after migration away from the sites of local delivery.nnnCONCLUSIONnThe active engraftment of the MSCs in combination with their previously documented immunomodulatory capabilities suggests the potential for therapeutic benefit in using MSCs for the treatment of periocular and joint diseases with immune involvement.

Dexamethasone Stiffens Trabecular Meshwork, Trabecular Meshwork Cells, and Matrix

PURPOSEnTreatment with corticosteroids can result in ocular hypertension and may lead to the development of steroid-induced glaucoma. The extent to which biomechanical changes in trabecular meshwork (TM) cells and extracellular matrix (ECM) contribute toward this dysfunction is poorly understood.nnnMETHODSnPrimary human TM (HTM) cells were cultured for either 3 days or 4 weeks in the presence or absence of dexamethasone (DEX), and cell mechanics, matrix mechanics and proteomics were determined, respectively. Adult rabbits were treated topically with either 0.1% DEX or vehicle over 3 weeks, and mechanics of the TM were determined.nnnRESULTSnTreatment with DEX for 3 days resulted in a 2-fold increase in HTM cell stiffness, and this correlated with activation of extracellular signal-related kinase 1/2 (ERK1/2) and overexpression of α-smooth muscle actin (αSMA). Further, the matrix deposited by HTM cells chronically treated with DEX is approximately 4-fold stiffer, more organized, and has elevated expression of matrix proteins commonly implicated in glaucoma (decorin, myocilin, fibrillin, secreted frizzle-related protein [SFRP1], matrix-gla). Also, DEX treatment resulted in a 3.5-fold increase in stiffness of the rabbit TM.nnnDISCUSSIONnThis integrated approach clearly demonstrates that DEX treatment increases TM cell stiffness concurrent with elevated αSMA expression and activation of the mitogen-activated protein kinase (MAPK) pathway, stiffens the ECM in vitro along with upregulation of Wnt antagonists and fibrotic markers embedded in a more organized matrix, and increases the stiffness of TM tissues in vivo. These results demonstrate glucocorticoid treatment can initiate the biophysical alteration associated with increased resistance to aqueous humor outflow and the resultant increase in IOP.

Safety and immunomodulatory effects of allogeneic canine adipose-derived mesenchymal stromal cells transplanted into the region of the lacrimal gland, the gland of the third eyelid and the knee joint.

BACKGROUND AIMSnMesenchymal stromal cells (MSCs) have been extensively studied as a cellular therapeutic for various pathologic conditions. However, there remains a paucity of data regarding regional and systemic safety of MSC transplantations, particularly with multiple deliveries of allogeneic cells. The purpose of this study was to investigate the safety and systemic immunomodulatory effects of repeated local delivery of allogeneic MSCs into the region of the lacrimal gland, the gland of the third eyelid and the knee joint in dogs.nnnMETHODSnAllogeneic adipose tissue-derived canine MSCs were delivered to the regions of the lacrimal gland and the third eyelid gland as well as in the knee joints of six healthy laboratory beagles as follows: six times with 1-week intervals for delivery to the lacrimal gland and the third eyelid gland regions and three to four times with 1- to 2-week intervals for intra-articular transplantations. Dogs were sequentially evaluated by clinical examination. At the conclusion of the study, dogs were humanely euthanized, and a complete gross and histopathologic examination of all organ systems was performed. Mixed leukocyte reactions were also performed before the first transplantation and after the final transplantation.nnnRESULTSnClinical and pathologic examinations found no severe consequences after repeated MSC transplantations. Results of mixed leukocyte reactions demonstrated suppression of T-cell proliferation after MSC transplantations.nnnCONCLUSIONSnThis is the first study to demonstrate regional and systemic safety and systemic immunomodulatory effects of repeated local delivery of allogeneic MSCs in vivo.

full-thickness Splinted Skin Wound Healing Models In Db/db And Heterozygous Mice: Implications For Wound Healing Impairment

The excisional dorsal full‐thickness skin wound model with or without splinting is widely utilized in wound healing studies using diabetic or normal mice. However, the effects of splinting on dermal wound healing have not been fully characterized, and there are limited data on the direct comparison of wound parameters in the splinted model between diabetic and normal mice. We compared full‐thickness excisional dermal wound healing in db/db and heterozygous mice by investigating the effects of splinting, semi‐occlusive dressing, and poly(ethylene glycol) treatment. Two 8‐mm full‐thickness wounds were made with or without splinting in db/db and heterozygous mice. Body weights, splint maintenance, wound contraction, wound closure, and histopathological parameters including reepithelialization, wound bed collagen deposition, and inflammation were compared between groups. Our results show that silicone splint application effectively reduced wound contraction in heterozygous and db/db mice. Splinted wounds, as opposed to nonsplinted wounds, exhibited no significant differences in wound closure between heterozygous and db/db mice. Finally, polyethylene glycol and the noncontact dressing had no significant effect on wound healing in heterozygous or db/db mice. We believe these findings will help investigators in selection of the appropriate wound model and data interpretation with fully defined parameters.

Importance of defining experimental conditions in a mouse excisional wound model.

The murine dorsum dermal excisional wound model has been widely utilized with or without splint application. However, variations in experimental methods create challenges for direct comparison of results provided in the literature and for design of new wound healing studies. Here, we investigated the effects of wound location and size, number of wounds, type of adhesive used for splint fixation on wound healing using splinted or unsplinted dorsum excisional full thickness wound models. One or two 6‐ or 8‐mm full thickness wounds were made with or without splinting in genetically diabetic but heterozygous mice (Dock7mu2009+u2009/u2009+u2009Leprdb). Two different adhesives: tissue adhesive and an over the counter cyanoacrylate adhesive (OTCA) “Krazy glue” were used to fix splints. Wound contraction, wound closure, and histopathological parameters including reepithelialization, collagen deposition and inflammation were compared between groups. No significant effect of wound number (1 vs. 2), side (left vs. right and cranial vs. caudal) or size on wound healing was observed. The OTCA group had a significantly higher splint success compared to the tissue adhesive group that resulted in significantly higher reepithelialization and collagen deposition in the OTCA group. Understanding the outcomes and effects of the variables will help investigators choose appropriate experimental conditions for the study purpose and interpret data.

Computed tomographic imaging characteristics of the normal canine lacrimal glands

BackgroundThe canine lacrimal gland (LG) and accessory lacrimal gland of the third eyelid (TEG) are responsible for production of the aqueous portion of the precorneal tear film. Immune-mediated, toxic, neoplastic, or infectious processes can affect the glands directly or can involve adjacent tissues, with secondary gland involvement. Disease affecting these glands can cause keratoconjunctivitis sicca, corneal ulcers, and loss of vision. Due to their location in the orbit, these small structures are difficult to evaluate and measure, making cross-sectional imaging an important diagnostic tool. The detailed cross-sectional imaging appearance of the LG and TEG in dogs using computed tomography (CT) has not been reported to date.ResultsForty-two dogs were imaged, and the length, width, and height were measured and the volume calculated for the LGs & TEGs. The glands were best visualized in contrast-enhanced CT images. The mean volume of the LG was 0.14xa0cm3 and the TEG was 0.1xa0cm3. The mean height, width, and length of the LG were, 9.36xa0mm, 4.29xa0mm, and 9.35xa0mm, respectively; the corresponding values for the TEG was 2.02xa0mm, 9.34xa0mm, and 7.90xa0mm. LG and TEG volume were positively correlated with body weight (pu2009<u20090.05).ConclusionsContrast-enhanced CT is a valuable tool for noninvasive assessment of canine lacrimal glands.

Assessment of tear film osmolarity using the TearLab™ osmometer in normal dogs and dogs with keratoconjunctivitis sicca

OBJECTIVEnTo evaluate repeatability and reproducibility of tear osmolarity measured using the TearLab™ osmometer in normal dogs and to assess its diagnostic potential in dogs with keratoconjunctivitis sicca (KCS).nnnANIMALS STUDIEDnBeagle dogs; six normal and five with KCS.nnnPROCEDURESnTear osmolarity and Schirmer tear test-1 (STT-1) values were obtained at various times. Normal dogs were assessed for diurnal variation and repeatability and reproducibility of measurements. Dogs with KCS were evaluated before and after 5 months topical twice-daily therapy with 2% cyclosporine.nnnRESULTSnMean ± SD tear osmolarity (mOsm/L) was significantly higher in normal dogs (337.4 ± 16.2) than in dogs with KCS before therapy (306.2 ± 18.0; P < 0.0001), but not following therapy with 2% cyclosporine (330.5 ± 13.7; P = 1.00). Osmolarity readings lower than 325.5 mOsm/L were suggestive of KCS (84.8% sensitivity and 87.1% specificity). In normal dogs, tear osmolarity readings were stable during the daytime (P = 0.99). Repeated measurements revealed high variability and typically poor-to-moderate repeatability and reproducibility, although this was improved by taking three successive measurements at each session. Considering combined data from all dogs, a positive correlation existed between STT-1 and tear osmolarity measurements (Pearsons correlation test, P = 0.04, r = 0.62).nnnCONCLUSIONSnCanine tear osmolarity as determined by TearLab™ osmometer was variable, required multiple readings to be informative, and differed from values reported for humans. Dogs with KCS had a lower tear osmolarity than did normal dogs, and this increased following cyclosporine therapy.

Gross, histologic, and micro-computed tomographic anatomy of the lacrimal system of snakes

OBJECTIVEnTo describe the lacrimal system of snakes using contrast micro-computed tomography (micro-CT) with 3-dimensional reconstruction, fluorescein passage (Jones) testing, histology, and gross dissection.nnnANIMALS STUDIEDnOne royal python and 19 snake cadavers representing 10 species.nnnPROCEDURESnDirect observation following injection of fluorescein into the subspectacular space, micro-CT following injection of three contrast agents into the subspectacular space, gross dissection following injection of latex into the subspectacular space, and histopathology.nnnRESULTSnInjection of fluorescein confirmed patency, but not course of the lacrimal duct. Barium enabled clear visualization of the lacrimal duct, whereas two iodinated contrast agents proved inadequate. Collectively, micro-CT, anatomic dissections, and histology suggest tears are produced by a single, large, serous, retrobulbar gland, released into the subspectacular space via several ductules, and drained through a single punctum originating in the ventronasal subspectacular space, and the lacrimal duct, which takes one of three routes of variable tortuosity before opening into the oral cavity in close association with the opening of the duct of the vomeronasal organ.nnnCONCLUSIONSnThe ophidian lacrimal duct has a generally tortuous course, and the details of its anatomy are species-variable. The tortuous course of the duct likely predisposes snakes to duct occlusion and must be considered when planning medical and surgical interventions in snakes with pseudobuphthalmos and subspectacular abscessation.

ASSESSMENT OF PLATELET-DERIVED GROWTH FACTOR USING A SPLINTED FULL THICKNESS DERMAL WOUND MODEL IN BEARDED DRAGONS (POGONA VITTICEPS)

Abstract: u2003 Wounds in reptiles are a common reason for presentation to a veterinarian. At this time there is limited information on effective topical medications to aid in wound closure. The objectives of this study were to translate the splinted, full-thickness dermal wound model, validated in mice, to the bearded dragon (Pogona vitticeps) and to determine the effect of topical becaplermin (BP), a platelet-derived growth factor (0.01%), on the rate of wound closure. Ten bearded dragons were anesthetized and two full-thickness cutaneous wounds were made on the dorsum of each lizard. Encircling splints were applied surrounding each wound and subsequently covered by a semi-occlusive dressing. Five lizards had one wound treated with BP and the adjacent wound treated with a vehicle control. Five additional lizards had one wound treated with saline and the second wound treated with a vehicle control. Wounds were imaged daily, and the wound area was measured using digital image analysis. The change in percentage wound closure over 17 days and the time to 50% wound closure was compared among the four treatment groups. There was no significant difference in wound closure rates between BP-treated and saline-treated wounds or in the time to 50% wound closure between any treatments. Vehicle-treated wounds adjacent to saline-treated wounds closed significantly slower than did BP (P < 0.010), saline (P < 0.001), and vehicle-treated wounds adjacent to BP-treated wounds (P < 0.013). Our preliminary study indicates that the splinted wound model, with modifications, may be used to determine wound closure rates in bearded dragons. When compared with saline, BP did not have a significant effect on wound closure rates, while the vehicle alone delayed wound closure. Histologic analysis of experimentally created wounds throughout the wound healing process is needed to further evaluate the effects of these treatments on reptile dermal wound healing.

Effects of chemical restraint on electroretinograms recorded sequentially in awake, sedated, and anesthetized dogs

OBJECTIVEnTo quantitatively and qualitatively compare electroretinography (ERG) recordings in awake, sedated, and anesthetized dogs.nnnANIMALSnSix 6-month-old Beagles.nnnPROCEDURESnA brief ERG protocol for dogs was used. Following 1-minute and subsequent 5-minute dark adaptation, mixed rod-cone responses were recorded bilaterally with a handheld multispecies ERG device with dogs in each of 3 states of consciousness: awake, sedated (dexmedetomidine and butorphanol), and anesthetized (atropine and hydromorphone, followed by propofol and midazolam and anesthetic maintenance with isoflurane). Low- and high-frequency noise levels were quantified via Fourier analysis, and the effect of consciousness state on signal amplitude, implicit time, and noise was analyzed via repeated-measures ANOVA. In addition, 13 veterinary ophthalmologists who were unaware of the dogs consciousness states subjectively graded the ERG recording quality, and scores for each tracing were compared.nnnRESULTSnERG amplitudes were highest in awake dogs and lowest in anesthetized dogs. Implicit times were shortest in awake dogs and longest in anesthetized dogs. Differences in b-wave amplitudes and a-wave implicit times were significant. Neither low- nor high-frequency noise levels differed significantly among consciousness states. Furthermore, no significant differences were identified among observers scores assigned to ERG tracings.nnnCONCLUSIONS AND CLINICAL RELEVANCEnAnesthesia and sedation resulted in significant attenuation and delay of ERG responses in dogs. Chemical restraint of dogs had no consistently significant effect on low- or high-frequency noise levels or on observer perception of signal quality.