Postnatal neural precursor cell regions in the rostral subventricular zone, hippocampal subgranular zone and cerebellum of the dog (Canis lupus familiaris).

Identification of neural stem and progenitor cells (NPCs) in vitro and in vivo is essential to the use of developmental and disease models of neurogenesis. The dog is a valuable large animal model for multiple neurodegenerative diseases and is more closely matched to humans than rodents with respect to brain organization and complexity. It is therefore important to determine whether immunohistochemical markers associated with NPCs in humans and rodents are also appropriate for the dog. The NPC markers CD15, CD133, nestin, GFAP and phosphacan (DSD-1) were evaluated in situ in the canine rostral telencephalon, hippocampal dentate gyrus, and cerebellum at different postnatal time-points. Positive staining results were interpreted in the context of region and cellular morphology. Our results showed that neurospheres and cells within the rostral subventricular zone (SVZ), dentate gyrus subgranular zone (SGZ), and white matter tracts of the cerebellum were immunopositive for CD15, nestin and GFAP. Neurospheres and the cerebellum were immunonegative for CD133, whereas CD133 staining was present in the postnatal rostral SVZ. Anti-phosphacan antibody staining delineated the neurogenic niches of the rostral lateral ventricle SVZ and the hippocampal SGZ. Positive staining for phosphacan was also noted in white matter tracts of the cerebellum and within the Purkinje layer. Our results showed that in the dog these markers were associated with regions shown to be neurogenic in rodents and primates.

Avian Inflammatory Markers.

Inflammation represents a fundamental response to diverse diseases ranging from trauma and infection to immune-mediated disease and neoplasia. As such, inflammation can be a nonspecific finding but is valuable as an indicator of pathology that can itself lead to disease if left unchecked. This article focuses on inflammatory biomarkers that are available and clinically useful in avian species. Inflammatory biomarkers are identified via evaluation of whole blood and plasma and can be divided into acute and chronic, with varying degrees of specificity and sensitivity. Evaluation of multiple biomarkers may be necessary to identify subclinical disease.

Biodistribution and pharmacodynamics of recombinant human alpha-L-iduronidase (rhIDU) in mucopolysaccharidosis type I-affected cats following multiple intrathecal administrations.

The storage disorder mucopolysaccharidosis type I (MPS I) is caused by a deficiency in lysosomal α-L-iduronidase activity. The inability to degrade glycosaminoglycans (GAG) results in lysosomal accumulation and widespread tissue lesions. Many symptoms of MPS I are amenable to treatment with recombinant human α-L-iduronidase (rhIDU), however, peripherally administered rhIDU does not cross the blood-brain barrier and has no beneficial effects in the central nervous system (CNS). A feline model of MPS I was used to evaluate the CNS effects of rhIDU following repeated intrathecal (IT) administration. Twelve animals were randomized into four groups based on the time of euthanasia and tissue evaluation following three repeat IT administrations of 0.1 mg/kg rhIDU or placebo on Study Days 1, 4 or 5, and 9. Two days after the final IT injection, the mean tissue α-L-iduronidase (IDU) activity in the brains of the two treated animals were approximately 3-times higher (50.1 and 54.9 U/mg protein) than the activity found in normal cat brains (mean of 18.3 U/mg), and remained higher than untreated MPSI brain at 1 month (2.4 and 4.1 U/mg protein) before returning to near-baseline levels after 2 months. This activity corresponded with decreased brain GAG concentrations after 2 days (1.4 and 2.0 µg/mg) and 1 month (0.9 and 1.1 µg/mg) which approached levels observed in normal animals (0.7 µg/mg). Attenuation of GAG, gangliosides GM2 and GM3, and cholesterol reaccumulation was identified at both two days and one month following final IT injection. No adverse effects attributable to IT rhIDU administration were observed. IT rhIDU may be an effective means for providing enzyme replacement therapy for the central manifestations of MPS I.

Transplantation and magnetic resonance imaging of canine neural progenitor cell grafts in the postnatal dog brain.

Cellular transplantation in the form of bone marrow has been one of the primary treatments of many lysosomal storage diseases (LSDs). Although bone marrow transplantation can help central nervous system manifestations in some cases, it has little impact in many LSD patients. Canine models of neurogenetic LSDs provide the opportunity for modeling central nervous system transplantation strategies in brains that more closely approximate the size and architectural complexity of the brains of children. Canine olfactory bulb-derived neural progenitor cells (NPCs) isolated from dog brains were expanded ex vivo and implanted into the caudate nucleus/thalamus or cortex of allogeneic dogs. Canine olfactory bulb-derived NPCs labeled with micron-sized superparamagnetic iron oxide particles were detected by magnetic resonance imaging both in vivo and postmortem. Grafts expressed markers of NPCs (i.e. nestin and glial fibrillary acidic protein), but not the neuronal markers Map2ab or beta-tubulin III. The NPCs were from dogs with the LSD mucopolysaccharidosis VII, which is caused by a deficiency of beta-glucuronidase. When mucopolysaccharidosis VII canine olfactory bulb-NPCs that were genetically corrected with a lentivirus vector ex vivo were transplanted into mucopolysaccharidosis VII recipient brains, they were detected histologically by beta-glucuronidase expression in areas identified by antemortem magnetic resonance imaging tracking. These results demonstrate the potential for ex vivo stem cell-based gene therapy and noninvasive tracking of therapeutic grafts in vivo.

In vitro growth and differentiation of canine olfactory bulb-derived neural progenitor cells under variable culture conditions.

The dog serves as a large animal model for multiple neurologic diseases that may potentially benefit from neural progenitor cell (NPC) transplantation. In the adult brain, multipotent NPCs reside in the subventricular zone and its rostral and caudal extensions into the olfactory bulb and hippocampus. The olfactory bulb represents a surgically accessible site for obtaining cells for autologous NPC transplantation. To model conditions that would occur for ex vivo gene therapy in the postnatal brain, NPCs were isolated from the canine olfactory bulb, expanded ex vivo under different culture conditions, and compared quantitatively for growth and immunophenotype. Under standard growth conditions, canine olfactory bulb-derived NPCs (OB-cNPCs) could be expanded nearly 500-fold in the time evaluated. Canine OB-cNPCs grown on poly-d-lysine (PDL) or on PDL-fibronectin had similar growth rates, whereas supplementation with leukemia inhibitory factor (LIF) resulted in significantly slower growth. However, when OB-cNPC cultures were grown on PDL-fibronectin or PDL supplemented with LIF, a greater proportion of cells with neuronal markers were generated upon differentiation.

Abnormalities in neural progenitor cells in a dog model of lysosomal storage disease.

Lysosomal storage disorders constitute a large group of genetic diseases, many of which are characterized by mental retardation and other neurologic symptoms. The mechanisms of neural dysfunction remain poorly understood. Because neural progenitor cells (NPCs) are fundamentally important to normal brain development and function, we investigated NPC properties in a canine model of mucopolysaccharidosis VII (MPS VII). MPS VII is a lysosomal storage disorder characterized by defects in the catabolism of glycosaminoglycans. NPCs were isolated from the olfactory bulb, cerebellum, and striatal subventricular zone of normal and MPS VII (beta-glucuronidase-deficient) postnatal dog brains. Canine NPCs (cNPCs) from normal and MPS VII brains had similar growth curves, but cerebellar-derived cNPCs grew significantly slower than those derived from other regions. In differentiation assays, MPS VII cNPCs from the striatal subventricular zone and cerebellum generated fewer mature neuronal and/or glial cells than normal, and MPS VII olfactory bulb-derived cNPCs retained significantly more phenotypically immature cells. These differences were only present at the earliest time point after isolation; at later passages, there were no differences attributable to genotype. The data suggest that MPS VII cNPCs respond differently to developmental cues in vivo, probably because of the diseased neural microenvironment rather than intrinsic cellular deficits.

Structure-specific patterns of neural stem cell engraftment after transplantation in the adult mouse brain.

Transplantation of neural stem cells (NSCs) may be useful for delivering exogenous gene products to the diseased CNS. When NSCs are transplanted into the developing mouse brain, they can migrate extensively and differentiate into cells appropriate to the sites of engraftment, in response to the normal signals directing endogenous cells to their appropriate fates. Much of the prior work on NSC migration in the adult brain has examined directed migration within or toward focal areas of injury such as ischemia, brain tumors, or 6-hydroxydopamine (6-OHDA) lesions. However, treatment of many genetic disorders that affect the CNS will require widespread dissemination of the donor cells in the postnatal brain, because the lesions are typically distributed globally. We therefore tested the ability of NSCs to migrate in the unlesioned adult mouse brain after stereotaxic transplantation into several structures including the cortex and hippocampus. NSC engraftment was monitored in live animals by magnetic resonance imaging (MRI) after superparamagnetic iron oxide (SPIO) labeling of cells. Histological studies demonstrated that the cells engrafted in significantly different patterns within different regions of the brain. In the cerebral cortex, donor cells migrated in all directions from the injection site. The cells maintained an immature phenotype and cortical migration was enhanced by trypsin treatment of the cells, indicating a role for cell surface proteins. In the hippocampus, overall cell survival and migration were lower but there was evidence of neuronal differentiation. In the thalamus, the transplanted cells remained in a consolidated mass at the site of injection. These variations in pattern of engraftment should be taken into account when designing treatment approaches in nonlesion models of neurologic disease.

Suppurative, nonseptic polyarthropathy in dogs.

The goals of this study were to determine the historical, physical examination, and clinicopathologic findings in dogs with suppurative, nonseptic polyarthropathy and to identify concurrent disorders associated with this syndrome. Medical records of 52 dogs with cytologic evidence of suppurative inflammation in two or more joints were examined retrospectively. Age of dogs was 4.8 years (median, range: 0.5-12 years). There was no clear breed or sex predilection, but most were large-breed dogs (body weight > or = 20 kg [44.4 lbs] in 40/52). Body temperature was 103.0 degrees F (39.4 degrees C) (median, range: 100.0-105.9 degrees F), with 29 of 52 dogs having a body temperature > or = 103 degrees F (39.4 degrees C). Lameness was identified in 42 of 52 dogs. Erosive changes were found in only 1 of 37 dogs that had radiography performed. A clear underlying disease process was not identified in 34 of 52 dogs. Seven dogs had evidence of infectious or inflammatory processes at extra-articular sites; 4 dogs were diagnosed with systemic lupus erythematosus (SLE); 2 dogs had gastrointestinal disease; 2 dogs had been vaccinated within 1 month before onset of polyarthritis; 1 dog had cancer; 1 dog had polyarthritis and meningitis; and 1 dog had erosive polyarthritis. Of the 44 dogs tested, 25 had antibodies to Borrelia burgdorferi, detected by an ELISA assay, which was significantly greater than the general hospital population (P = .007). Antibodies against Rickettsia rickettsiae and Ehrlichia canis were not definitively identified in the sera of any dog tested in this study (45 and 44 dogs, respectively). We conclude that an underlying disease process is not identified in most cases of suppurative polyarthropathy in dogs and that intestinal disease, neoplasia, and SLE are uncommon causes of polyarthritis. While seropositivity against the causative agent of Lyme disease was common and possibly a cause of polyarthritis in some dogs of our study, evidence of other vector-borne infection was not identified.

The Effects of Acute Blood Loss for Diagnostic Bloodwork and Fluid Replacement in Clinically Ill Mice.

Despite the great value of diagnostic bloodwork for identifying disease in animals, the volume of blood required for these analyses limits its use in laboratory mice, particularly when they are clinically ill. We sought to determine the effects of acute blood loss (ABL) following blood collection for diagnostic bloodwork in healthy mice compared with streptozotocin-induced diabetic and dextran sulfate sodium (DSS)-treated dehydrated mice. ABL caused several mild changes in the control mice, with significant decreases in body weight, temperature, and activity in both experimental groups; increased dehydration and azotemia in the DSS-treated mice; and a significant drop in the blood pressure of the diabetic mice. To determine whether these negative outcomes could be ameliorated, we treated mice with intraperitoneal lactated Ringers solution either immediately after or 30 min before ABL. Notably, preABL administration of fluids helped prevent the worsening of the dehydration and azotemia in the DSS-treated mice and the changes in blood pressure in the diabetic mice. However, fluid administration provided no benefit in control of blood pressure when administered after ABL in the diabetic mice. Furthermore, fluid therapy did not prevent ABL-induced drops in body weight and activity. Although one mouse not receiving fluid therapy became moribund at the 24-h time point, no animals died during the 24-h study. This investigation demonstrates that blood for diagnostic bloodwork can be collected safely from clinically ill mice and that preemptive fluid therapy mitigates some of the negative changes associated with this blood loss.

Gelatinase activity in synovial fluid and synovium obtained from healthy and osteoarthritic joints of dogs.

OBJECTIVE: To determine matrix metalloproteinase (MMP) activity in synovial fluid (SF) obtained from the joints of dogs with degenerative joint disease (DJD) secondary to various underlying conditions. SAMPLE POPULATION: 35 samples of SF obtained from 18 clinically normal (control) dogs and 34 samples of SF obtained from 17 dogs with DJD; dogs with DJD were from 2 populations (client-owned dogs and research dogs that had DJD secondary to the lysosomal storage disease mucopolysaccharidosis VII). PROCEDURE: MMP activity in samples of SF was semiquantitatively examined by use of gelatin or casein zymography. Western blot analysis was performed by use of antibodies for MMP-2 and MMP-9. In addition, in situ MMP activity was observed in sections of synovial membrane obtained from healthy and osteoarthritic joints. RESULTS: Samples of SF from osteoarthritic joints had higher MMP-2 activity and dramatically increased MMP-9 activity, compared with values for healthy joints. Substrate-overlay analyses indicated minimal gelatin-degrading activity in synoviocytes obtained from control dogs, whereas greater activity was seen in osteoarthritic synoviocytes, with additional activity in the underlying tissue. CONCLUSIONS AND CLINICAL RELEVANCE: Higher MMP-2 activity and dramatic increases in MMP-9 activity were associated with the osteoarthritic state, even though MMP-2 activity was detected in healthy joints. This study expands information on MMP production in SF of osteoarthritic joints in other species and documents the similarity of MMP activity patterns regardless of the cause of DJD.

Significance of mastocytemia in cats.

BACKGROUND: In cats, mastocytemia is considered to be confined to animals with mast cell tumors (MCT), whereas in dogs it is associated with diverse diseases. OBJECTIVE: The objective of this retrospective study was to investigate the diagnostic and prognostic significance of mastocytemia in cats. METHODS: All blood smears and buffy coat (BC) smears on which mast cells were identified over a 6-year period were retrospectively reviewed and mast cells counted. Mastocytemic cats were classified based on their clinical diagnosis. RESULTS: Mastocytemia was identified on 40 blood smears and 13 BC smears from 33 cats. The incidence of mastocytemia detected in cats during routine CBCs was 0.33% (40/12,116 CBCs). Twenty-two of 33 mastocytemic cats (67%) had visceral (n = 17) or cutaneous MCT (n = 7), including 2 that had concurrent visceral and cutaneous involvement. In 3 additional cases (9%), visceral MCT was clinically suspected, but no cytologic or histopathologic evaluation of visceral organs was performed. MCT was excluded in 3 of 33 mastocytemic cats (9%) with a final diagnosis of lymphoid neoplasia (n = 2) and multiorgan hemangiosarcoma (n = 1). Five additional animals (15%) had a diagnosis other than MCT, including lymphoma (n = 2) and chronic renal failure (n = 3), but no cytologic or histopathologic evaluation of the spleen was performed. Blood smears from cats with confirmed MCT had 1-113 mast cells per smear, whereas cats in which MCT was excluded had 1-2 mast cells per smear. CONCLUSIONS: Data confirm that mastocytemia is rare and most commonly found in cats with visceral MCT; however, rare circulating mast cells may also be seen with neoplasms other than MCT.

Subject-based reference values: biological variation, individuality, and reference change values.

Subject-based reference values have been largely overlooked in veterinary medicine. These values represent longitudinal data rather than the cross-sectional data represented by standard population-based reference values. As such they provide information about biological and analytical variation. Inherent random variation of analytes around a homeostatic set point is referred to as biological variation; data on biological variation are underutilized in veterinary medicine and have multiple applications that include setting analytical goals, predicting the utility of population-based reference intervals (RIs), assessing the value of partitioning reference values, and evaluating the significance of changes in serial results. To generate these data, relatively few individuals are sampled for a short period of time. Given the difficulty of obtaining specimens from large number of healthy individuals to establish a cross-sectional RI for many veterinary species, especially exotic species, use of subject-based RIs is a practical alternative approach for the veterinary diagnostician. Furthermore, for the majority of biochemical analytes and even many hemostatic variables, population-based reference values are less sensitive than subject-based reference values for detecting pathologic changes in an individual. The focus of this review is the clinical usefulness of subject-based reference values and diagnostic implications for their use. Implementation of the concepts of biological variation, individuality, and reference change value (RCV) may allow large diagnostic laboratories to offer more sensitive reference values to assess health and detect disease.

ASVCP reference interval guidelines: determination of de novo reference intervals in veterinary species and other related topics.

Reference intervals (RI) are an integral component of laboratory diagnostic testing and clinical decision-making and represent estimated distributions of reference values (RV) from healthy populations of comparable individuals. Because decisions to pursue diagnoses or initiate treatment are often based on values falling outside RI, the collection and analysis of RV should be approached with diligence. This report is a condensation of the ASVCP 2011 consensus guidelines for determination of de novo RI in veterinary species, which mirror the 2008 Clinical Laboratory and Standards Institute (CLSI) recommendations, but with language and examples specific to veterinary species. Newer topics include robust methods for calculating RI from small sample sizes and procedures for outlier detection adapted to data quality. Because collecting sufficient reference samples is challenging, this document also provides recommendations for determining multicenter RI and for transference and validation of RI from other sources (eg, manufacturers). Advice for use and interpretation of subject-based RI is included, as these RI are an alternative to population-based RI when sample size or inter-individual variation is high. Finally, generation of decision limits, which distinguish between populations according to a predefined query (eg, diseased or non-diseased), is described. Adoption of these guidelines by the entire veterinary community will improve communication and dissemination of expected clinical laboratory values in a variety of animal species and will provide a template for publications on RI. This and other reports from the Quality Assurance and Laboratory Standards (QALS) committee are intended to promote quality laboratory practices in laboratories serving both clinical and research veterinarians.

Malignant pilomatricoma in a soft-coated Wheaten Terrier.

A 3-year-old, castrated male, soft-coated Wheaten Terrier was presented for evaluation of mild lameness, fecal incontinence, lumbosacral pain, and lack of anal tone. Magnetic resonance imaging scan showed a large (8 x 6 x 5 cm) mass invading and expanding the pelvic bones, sacrum, and associated structures. A fine-needle aspirate of the mass contained many neoplastic cells with high nuclear to cytoplasmic ratios and rare spindle and inflammatory cells. The neoplastic cells were 12-16 mum in diameter, round to cuboidal, basaloid in appearance, and arranged both individually and in loosely cohesive clusters with variably distinct cell borders. Given the location, signalment, and cytologic findings, differential interpretations included a primitive embryonal tumor (eg, neuroblastoma or nephroblastoma in an atypical location) or poorly differentiated carcinoma. The owner elected euthanasia due to the poor prognosis. Abnormal gross findings on necropsy included the pelvic mass and multiple firm, pale, pink-tan nodules in the lung, which proved to be metastases. On histologic examination, the mass and nodules were composed of irregular islands, lobules, and nests of basaloid cells, which transitioned abruptly into large lakes of "ghost" cells with areas of ossification and calcification, consistent with a diagnosis of malignant pilomatricoma. This unusual presentation of a pilomatricoma adds to our knowledge of expected cytologic findings for this tumor.

Hepatic and metabolic changes in surgical colic patients: a pilot study.

OBJECTIVE: To determine: (1) changes in blood ammonia, bile acid (BA), bilirubin, triglyceride, and glucose concentrations and liver enzyme activities in perioperative colic patients and (2) the association between these laboratory findings and short-term survival. DESIGN: Prospective observational clinical study. ANIMALS: Thirty-two adult horses undergoing exploratory celiotomy for colic. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Blood samples were collected preoperatively and at 24-36 and 72-84 hours postoperatively and analyzed for blood ammonia, BA, bilirubin, triglyceride, and glucose concentrations and sorbitol dehydrogenase (SDH) and gamma glutamyl transferase (GGT) activities. Short-term survival was defined as survival to hospital discharge. Data were analyzed using a Fisher's exact test and analysis of variance. Mildly increased blood ammonia concentrations were present in 2 horses at admission. Postoperative blood ammonia concentrations were within reference intervals in all horses. There were increases in liver enzyme activities as well as in BA, triglyceride, and total bilirubin concentrations. Horses with markedly increased admission BA concentrations and SDH activities did not survive. BA concentrations and SDH activities decreased postoperatively. There was no association between GGT activity and survival; GGT activity remained increased postoperatively. Blood triglyceride concentration was increased in almost all horses postoperatively; horses that did not survive had higher triglyceride concentrations at 24-36 hours postoperatively than horses that survived. CONCLUSION: Alterations in metabolism and hepatobiliary function are common in colic patients. The results of this study provide further prognostic indices for colic patients and highlight areas for improvement in patient management.

Magnetic resonance imaging detects differences in migration between primary and immortalized neural stem cells.

RATIONALE AND OBJECTIVES: The study was performed to evaluate the effect of magnetic resonance imaging (MRI) contrast agent (super paramagnetic iron oxide [SPIO]) on differentiation and migration of primary murine neural stem cells (NSCs) in comparison to a neural stem cell line (C17.2). Because detection of labeled cells depends on the concentration of SPIO particles per imaging voxel, the study was performed at various concentrations of SPIO particles to determine the concentration that could be used for in vivo detection of small clusters of grafted cells. MATERIALS AND METHODS: Murine primary NSCs or C17.2 cells were labeled with different concentrations of SPIO particles (0, 25, 100, and 250 microg Fe/mL) and in vitro assays were performed to assess cell differentiation. In vivo MRI was performed 7 weeks after neonatal transplantation of labeled cells to evaluate the difference in migration capability of the two cell populations. RESULTS: Both the primary NSCs and the C17.2 cells differentiated to similar number of neurons (Map2ab-positive cells). Similar patterns of engraftment of C17.2 cells were seen in transplanted mice regardless of the SPIO concentration used. In vivo MRI detection of grafted primary and C17.2 cells was only possible when cells were incubated with 100 microg/mL or higher concentration of SPIO. Extensive migration of C17.2 cells throughout the brain was observed, whereas the migration of the primary NSCs was more restricted. CONCLUSIONS: Engraftment of primary NSCs can be detected noninvasively by in vivo MRI, and the presence of SPIO particles do not affect the viability, differentiation, or engraftment pattern of the donor cells.