Judith B. Koenig

Evaluation of ultrasmall superparamagnetic iron oxide contrast agent labeling of equine cord blood and bone marrow mesenchymal stromal cells

OBJECTIVE To evaluate the efficacy and effects of labeling equine umbilical cord blood (UCB)- and bone marrow (BM)-derived multipotent mesenchymal stromal cells (MSCs) with an ultrasmall superparamagnetic iron oxide (SPIO) contrast agent and the detection of labeled MSCs by use of MRI. SAMPLE UCB MSCs from placental tissues of 5 foals and BM MSCs from 5 horses. PROCEDURES UCB and BM MSC cultures were seeded in duplicate (5,000 cells/cm(2)). One duplicate was incubated with SPIO (50 μg/mL); the other was processed identically, but without SPIO. Mesenchymal stromal cells were expanded in triplicates for 5 passages and assessed for viability and proliferative capacity, labeling efficacy, and labeled cell proportion. For MRI detection, 5 × 10(6) labeled BM MSCs from passage 1 or 2 were injected into a collagenase-induced superficial digital flexor tendon defect of an equine cadaveric forelimb from 2 horses. RESULTS For passages 1, 2, and 3, labeling efficacy and cell proportion for UCB MSCs (99.6% [range, 98.8% to 99.9%], 16.6% [range, 6.5% to 36.1%], and 1.0% [range, 0.4% to 2.8%], respectively) were significantly higher than for BM MSCs (99.2% [range, 97.8% to 99.7%], 4.5% [range, 1.6% to 11.8%], and 0.2% [range, 0.1% to 0.6%], respectively). Labeling was not detectable after passage 3. Viability of MSCs was not affected, but cell doubling time increased in labeled MSCs, compared with that of unlabeled MSCs. On MRI 3-D T2*-weighted fast gradient echo sequences, decreased signal intensity was observed for BM passage 1 MSCs. CONCLUSIONS AND CLINICAL RELEVANCE Equine UCB and BM MSCs were labeled with SPIO at high efficiencies.

Effects of unfocused extracorporeal shock wave therapy on healing of wounds of the distal portion of the forelimb in horses

OBJECTIVE To determine effects of extracorporeal shock wave therapy (ESWT) on healing of wounds in the distal portion of the forelimb in horses. ANIMALS 6 horses. PROCEDURES Five 6.25-cm2 superficial wounds were created over both third metacarpi of 6 horses. Forelimbs were randomly assigned to treatment (ESWT and bandage) or control (bandage only) groups. In treated limbs, each wound was treated with 625 shock wave pulses from an unfocused electrohydraulic shock wave generator. In control limbs, each wound received sham treatment. Wound appearance was recorded weekly as inflamed or healthy and scored for the amount of protruding granulation tissue. Standardized digital photographs were used to determine the area of neoepithelialization and absolute wound area. Biopsy was performed on 1 wound on each limb every week for 6 weeks to evaluate epithelialization, fibroplasia, neovascularization, and inflammation. Immunohistochemical staining for A smooth muscle actin was used to label myofibroblasts. RESULTS Control wounds were 1.9 times as likely to appear inflamed, compared with treated wounds. Control wounds had significantly higher scores for exuberant granulation tissue. Treatment did not affect wound size or area of neoepithelialization. No significant difference was found for any of the histologic or immunohistochemical variables between groups. CONCLUSIONS AND CLINICAL RELEVANCE Treatment with ESWT did not accelerate healing of equine distal limb wounds, but treated wounds had less exuberant granulation tissue and appeared healthier than controls. Therefore, ESWT may be useful to prevent exuberant granulation tissue formation and chronic inflammation of such wounds, but further studies are necessary before recommending ESWT for clinical application.

Evaluation of endotoxin activity in blood measured via neutrophil chemiluminescence in healthy horses and horses with colic

OBJECTIVE To evaluate the performance of a chemiluminescent endotoxin activity assay in horses with colic and healthy horses. ANIMALS 20 horses with colic and systemic inflammatory response syndrome (SIRS group), 8 horses with colic with no SIRS (NSIRS group), and 20 healthy horses. PROCEDURES Venous blood was collected into EDTA blood collection tubes after completion of a physical examination, and a chemiluminescent endotoxin activity assay was performed within 60 minutes of collection. Medical or surgical interventions and outcome were recorded for each horse. RESULTS Mean + or - SE endotoxin activity was 0.16 + or - 0.05 for healthy horses, 0.18 + or - 0.07 for the NSIRS group, and 0.53 + or - 0.05 for the SIRS group and was significantly different among the groups. Mean endotoxin activity was significantly higher in the SIRS group than in the NSIRS group and the healthy group. No significant difference between the healthy and NSIRS groups was present. The higher the measured endotoxin activity, the more likely it was for horses to be euthanized. CONCLUSIONS AND CLINICAL RELEVANCE The chemiluminescent endotoxin assay was easy to use, required a short time to perform, could be completed at the patients side, and with some modifications, may be a useful component in the clinical assessment and prognostication of horses with colic.

Use of multichannel electrogastrography for noninvasive assessment of gastric myoelectrical activity in dogs.

OBJECTIVE To evaluate whether changes in gastric myoelectrical activity in healthy, awake dogs can be detected via multichannel electrogastrography (EGG). ANIMALS 6 healthy hound-breed dogs. PROCEDURES For each dog, 8-channel EGG was performed after food had been withheld for 12 hours and at 30 minutes after subsequent feeding; 60 minutes after feeding, atropine (0.04 mg/kg) was administered IM to induce ileus, and 30 minutes later, EGG was again performed. Mean cycles per minute (cpm) values of the dominant frequency (a measure of the rhythmicity of gastric electrical activity) and mean power ratios (ie, power measured after treatment divided by the power measured when food was withheld) were calculated. Motility of the gastric antrum was assessed via B-mode ultrasonography during the same phases; contractions determined ultrasonographically were correlated with EGG power for each channel in each phase. RESULTS The criterion for stability (SD of the dominant frequency < 15% of the cpm value in at least 3 of the 8 EGG channels) was met in 4 of the 6 dogs (only in long-distance channels). The mean power ratios were significantly higher in the postprandial phase than in the ileus phase. Compared with the postprandial phase, significantly fewer contractions per minute were evident ultrasonographically in the ileus and food-withholding phases. There was a significant and good correlation between EGG power and ultrasonographic findings in all 8 channels. CONCLUSIONS AND CLINICAL RELEVANCE Electrogastrography may be useful in assessing gastric myoelectrical activities in awake dogs with naturally occurring gastrointestinal disease, including gastric dilatation-volvulus.

Response to Intravenous Allogeneic Equine Cord Blood-Derived Mesenchymal Stromal Cells Administered from Chilled or Frozen State in Serum and Protein-Free Media

Equine mesenchymal stromal cells (MSC) are commonly transported, chilled or frozen, to veterinary clinics. These MSC must remain viable and minimally affected by culture, transport, or injection processes. The safety of two carrier solutions developed for optimal viability and excipient use were evaluated in ponies, with and without allogeneic cord blood-derived (CB) MSC. We hypothesized that neither the carrier solutions nor CB-MSC would elicit measurable changes in clinical, hematological, or biochemical parameters. In nine ponies (study 1), a bolus of HypoThermosol® FRS (HTS-FRS), CryoStor® CS10 (CS10), or saline was injected IV (n = 3/treatment). Study 2, following a 1-week washout period, 5 × 107 pooled allogeneic CB-MSCs were administered IV in HTS-FRS following 24 h simulated chilled transport. Study 3, following another 1-week washout period 5 × 107 pooled allogeneic CB-MSCs were administered IV in CS10 immediately after thawing. Nine ponies received CB-MSCs in study 2 and 3, and three ponies received the cell carrier media without cells. CB-MSCs were pooled in equal numbers from five unrelated donors. In all studies, ponies were monitored with physical examination, and blood collection for 7 days following injection. CD4 and CD8 lymphocyte populations were also evaluated in each blood sample. In all three studies, physical exam, complete blood cell count, serum biochemistry, and coagulation panel did not deviate from established normal ranges. Proportions of CD4+ and CD8+ lymphocytes increased at 168 h postinjection in CB-MSC treatment groups regardless of the carrier solution. Decreases in CD4+/CD8+ double positive populations were observed at 24 and 72 h in CB-MSC-treated animals. There was no difference in viability between CB-MSCs suspended in HTS-FRS and CS10. HTS-FRS and CS10 used for low volume excipient injection of MSC suspensions were not associated with short-term adverse reactions. HTS-FRS and CS10 both adequately maintain CB-MSC viability following hypothermic or frozen simulated transport, respectively. CB-MSCs do not elicit clinical abnormalities, but allogeneic stimulation of CD4+ and CD8+ lymphocyte populations may occur. Future studies should include in vitro or in vivo evaluation of cell-mediated or adaptive immunity to autologous, identical allogeneic, or MSC originating from additional unrelated individuals in order to better characterize this response.

Comparison of laparoscopic versus conventional open cryptorchidectomies on intraoperative and postoperative complications and duration of surgery, anesthesia, and hospital stay in horses

Objective-To compare surgical preparation time, surgery and anesthesia times, hospitalization duration, and intra- and postoperative complications between laparoscopic and conventional open cryptorchidectomy in horses. Design-Retrospective cohort study. Animals-60 horses that underwent cryptorchidectomy. Procedures-Medical records were reviewed to identify horses that had undergone cryptorchidectomy from 1991 to 2012. Thirty horses that underwent laparoscopic cryptorchidectomy (case horses) were matched with 30 control horses that had undergone open cryptorchidectomy (ie, inguinal and parainguinal surgical approaches). Horses were matched according to history of previous surgery, testicle location, and type of closure following removal of an undescended unilateral testicle. Duration of surgery, surgical preparation and anesthesia times, hospitalization duration, and number of intra- and postoperative complications were compared between horses that underwent laparoscopic cryptorchidectomy versus open cryptorchidectomy. Comparisons were also made between horses in terms of whether there was a history of previous failed cryptorchidectomy or unknown location of testicle prior to surgery. Results-Horses that underwent laparoscopic cryptorchidectomy had significantly longer surgery and anesthesia times overall, compared with horses that underwent open cryptorchidectomy. No difference in surgery time was found between case and control horses that had a previous surgical attempt to remove an undescended testicle or in which the testicle location was unknown prior to surgery. Overall, horses undergoing laparoscopy had a nonsignificant increase in intraoperative complications, compared with control horses, and had significantly more postoperative complications. Conclusions and Clinical Relevance-Horses undergoing laparoscopic cryptorchidectomy had increased surgical preparation time, increased surgery and anesthesia times, and more postoperative complications, compared with horses undergoing open cryptorchictomy. Laparoscopy may be advantageous for a second attempt at cryptorchidectomy or if the testicle location is unknown prior to surgery.

Use of multichannel electrointestinography for noninvasive assessment of myoelectrical activity in the cecum and large colon of horses

OBJECTIVE To evaluate whether changes in myoelectrical activity in the cecum and large colon of horses can be detected via multichannel electrointestinography (EIG). ANIMALS 6 healthy mature horses. PROCEDURES Each horse underwent 3 EIG procedures. Intestinal myoelectrical activity (cecum and large colon) was recorded during a 20-minute period following i.v. administration of physiologic saline (0.9% NaCl) solution (20 mL; baseline), erythromycin lactobionate (0.5 mg/kg), or detomidine (0.015 mg/kg); intestinal contractions were concurrently viewed via B-mode ultrasonography. By use of computer software, 8-channel EIG recordings were analyzed and the mean of the dominant frequency (a measure of the rhythmicity of gastric electrical activity) expressed in cycles per minute (cpm) was obtained. Total power (muV(2)) was calculated, and treatment effect was expressed as the power ratio (ie, treatment-associated power divided by the baseline power). RESULTS The dominant frequency cpm values were not stable, and no significant differences between treatments were detected. Compared with the effects of saline solution treatment, detomidine significantly reduced the mean cecal and colonic power ratios. Erythromycin significantly reduced the cecal power ratio and increased the colonic power ratio, although the increase was significant in only 1 channel. Ultrasonographic findings and total power (predominantly from the long-distance electrode pairs) were significantly correlated. CONCLUSIONS AND CLINICAL RELEVANCE In horses, EIG was useful for assessment of changes in myoelectrical activity in the cecum and large colon. Multiple electrodes should be used to cover a larger area of the intestine, and agreement between multiple channels is needed to make the analysis meaningful.

Post-Thaw Non-Cultured and Post-Thaw Cultured Equine Cord Blood Mesenchymal Stromal Cells Equally Suppress Lymphocyte Proliferation In Vitro

Multipotent mesenchymal stromal cells (MSC) are receiving increased attention for their non-progenitor immunomodulatory potential. Cryopreservation is commonly used for long-term storage of MSC. Post-thaw MSC proliferation is associated with a lag-phase in vitro. How this lag-phase affect MSC immunomodulatory properties is unknown. We hypothesized that in vitro there is no difference in lymphocyte suppression potential between quick-thawed cryopreserved equine cord blood (CB) MSC immediately included in mixed lymphocyte reaction (MLR) and same MSC allowed post-thaw culture time prior to inclusion in MLR. Cryopreserved CB-MSC from five unrelated foals were compared using two-way MLR. For each of the five unrelated MSC cultures, paired MLR assays of MSC allowed five days of post-thaw culture and MSC included in MLR assay immediately post-thawing were evaluated. We report no difference in the suppression of lymphocyte proliferation by CB-MSC that had undergone post-thaw culture and MSC not cultured post-thaw (p<0.0001). Also, there was no inter-donor variability between the lymphocyte suppressive properties of MSC harvested from the five different donors (p = 0.13). These findings suggest that cryopreserved CB-MSC may have clinical utility immediately upon thawing. One implication hereof is the possibility of using cryopreserved CB-MSC at third party locations without the need for cell culture equipment or competencies.

Anesthetic management of a white rhinoceros (Ceratotherium simum) undergoing an emergency exploratory celiotomy for colic

OBSERVATIONS A 26-year-old male white rhinoceros (Ceratotherium simum), weighing approximately 2000 kg was anesthetized for an exploratory celiotomy. Sedation was achieved with intramuscular butorphanol (0.04 mg kg(-1)) and detomidine (0.025 mg kg(-1)) and induction of anesthesia with intravenous glyceryl guaiacolate (50 g) and three intravenous boluses of ketamine (200 mg, each); the trachea was then intubated and anesthesia maintained with isoflurane in oxygen using a circle breathing system. Positioning in dorsal recumbency for the surgery and later in sternal recumbency for the recovery represented challenges that added to the prolonged anesthesia time and surgical approach to partially correct an impaction. The rhinoceros recovered uneventfully after 10.4 hours of recumbency. CONCLUSIONS Anesthetic management for an exploratory celiotomy with a midline approach is possible in rhinoceroses, although planning and extensive staff support is necessary to adequately position the patient.