Quantitative morphology in canine cutaneous soft tissue sarcomas

Stained cytological specimens from 24 dogs with spontaneous soft tissue sarcomas [fibrosarcoma (n = 8), liposarcoma (n = 8) and haemangiopericytoma (n = 8)], and 24 dogs with reactive connective tissue lesions [granulation tissue (n = 12) and dermal fibrosis (n = 12)] were analysed by computer‐assisted nuclear morphometry. The studied morphometric parameters were: mean nuclear area (MNA; µm²), mean nuclear perimeter (MNP; µm), mean nuclear diameter (MND mean; µm), minimum nuclear diameter (D_min; µm) and maximum nuclear diameter (D_max; µm). The study aimed to evaluate (1) possibility for quantitative differentiation of soft tissue sarcomas from reactive connective tissue lesions and (2) by using cytomorphometry, to differentiate the various histopathological soft tissue sarcomas subtypes in dogs. The mean values of all nuclear cytomorphometric parameters (except for D_max) were statistically significantly higher in reactive connective tissue processes than in soft tissue sarcomas. At the same time, however, there were no considerable differences among the different sarcoma subtypes. The results demonstrated that the quantitative differentiation of reactive connective tissue processes from soft tissue sarcomas in dogs is possible, but the same was not true for the different canine soft tissue sarcoma subtypes. Further investigations on this topic are necessary for thorough explication of the role of quantitative morphology in the diagnostics of mesenchymal neoplasms and tumour‐like fibrous lesions in dogs     

Intra-operative cisplatin for the treatment of canine extremity soft tissue sarcomas

Nineteen dogs with histologically confirmed soft tissue sarcomas of the extremities were treated with a combination of marginal surgery and intra-operative chemotherapy in the form of cisplatin in a biodegradable implant delivery system (Atrigel^®; Atrix Laboratories, Fort Collins, Co, USA). None of the dogs had evidence of metastasis at time of treatment. The median dose of cisplatin was 52.1 mg/m² (mean 55.4 mg/m², range 18.5–108.6 mg/m²). Wound complications were noted in 16 dogs (84.2%). Median follow-up time was 874 days (mean 777 days, range 125–1463 days). Nine dogs (47.3%) were alive at the time of analysis. Local recurrence occurred in three dogs (16.6%). The time to recurrence was 214, 264 and 874 days.     

Differentiation of canine bone marrow stromal cells into voltage- and glutamate-responsive neuron-like cells by basic fibroblast growth factor

We investigated the in vitro differentiation of canine bone marrow stromal cells (BMSCs) into voltage- and glutamate-responsive neuron-like cells. BMSCs were obtained from the bone marrow of healthy beagle dogs. Canine BMSCs were incubated with the basal medium for neurons containing recombinant human basic fibroblast growth factor (bFGF; 100 ng/ml). The viability of the bFGF-treated cells was assessed by a trypan blue exclusion assay, and the morphology was monitored. Real-time RT-PCR was performed to evaluate mRNA expression of neuronal, neural stem cell and glial markers. Western blotting and immunocytochemical analysis for the neuronal markers were performed to evaluate the protein expression and localization. The Ca²⁺ mobilization of the cells was evaluated using the Ca²⁺ indicator Fluo3 to monitor Ca²⁺ influx. To investigate the mechanism of bFGF-induced neuronal differentiation, the fibroblast growth factor receptor inhibitor, the phosphoinositide 3-kinase inhibitor or the Akt inhibitor was tested. The bFGF treatment resulted in the maintenance of the viability of canine BMSCs for 10 days, in the expression of neuronal marker mRNAs and proteins and in the manifestation of neuron-like morphology. Furthermore, in the bFGF-treated BMSCs, a high concentration of KCl and L-glutamate induced an increase in intracellular Ca²⁺ levels. Each inhibitor significantly attenuated the bFGF-induced increase in neuronal marker mRNA expression. These results suggest that bFGF contributes to the differentiation of canine BMSCs into voltage- and glutamate-responsive neuron-like cells and may lead to the development of new cell-based treatments for neuronal diseases.     

Serum fibroblast growth factor 23 (FGF-23): associations with hyperphosphatemia and clinical staging of feline chronic kidney disease

Fibroblast growth factor 23 (FGF-23) is an independent monitor of the progression of chronic kidney disease (CKD) in human medicine, and FGF-23 may have value as a biomarker in feline CKD. We evaluated the relationship between serum FGF-23 and CKD stages, and the effect of age on FGF-23 in normal cats. We measured FGF-23 and intact parathyroid hormone (iPTH) concentrations by ELISA, with intra- and inter-assay CVs ≤ 15%. The percentage recovery of FGF-23 and iPTH remained stable for up to 7 d in samples stored at −20°C and −80°C. We measured FGF-23 in 304 cats, among which 196 were diagnosed with CKD. The 108 clinically healthy cats were divided into 5 subgroups based on growth stage (0–2 y, 3–6 y, 7–10 y, 11–14 y, ≥ 15 y). No statistical difference was found in FGF-23 among age groups (p = 0.15) or by sex in healthy subjects. Using the International Renal Interest Society guideline, 34 cats were defined as CKD stage 1, 74 stage 2, 51 stage 3, and 37 stage 4. FGF-23 was higher in cats in all CKD stages than in controls. Higher serum phosphorus was observed in stage 3 (p = 0.04) and 4 (p < 0.01) compared to controls. iPTH increased as CKD progressed. Pearson analysis indicated a positive linear relationship between FGF-23 and iPTH (control: r = 0.70, p < 0.01; CKD: r = 0.46, p = 0.02). FGF-23 may be a useful biomarker of feline CKD and may precede hyperphosphatemia in advanced feline CKD.     

Diagnostic accuracy of pre‐treatment biopsy for grading soft tissue sarcomas in dogs

Histologic grade is an important prognostic factor for both local recurrence and metastatic potential with canine soft tissue sarcoma (STS). Pre‐treatment biopsy with identification of tumour grade may aid in prognostication and determination of surgical margins necessary for local control. The purpose of this study was to evaluate the grading accuracy of various pre‐treatment biopsy techniques (wedge, punch, needle‐core) for STS in dogs. Medical records of 68 dogs diagnosed with a STS via pre‐treatment biopsy and confirmed by excisional biopsy were evaluated. The concordance in grade between excisional and pre‐treatment biopsies was 59%. Of the 41% that lacked concordance, 29% of pre‐treatment biopsies underestimated and 12% overestimated grade. The method of pre‐treatment biopsy did not significantly effect grade concordance. Based on these data, needle‐core biopsy appears to be similar in accuracy compared to open biopsy, however, grading determined by pre‐treatment biopsy in general should be interpreted with caution.     

Expression of vascular endothelial growth factor in canine oral malignant melanoma

Serum, plasma and tissue expression of vascular endothelial growth factor (VEGF) was measured in 20 dogs previously diagnosed histologically with oral melanoma. The concentrations of VEGF in serum and plasma were significantly higher in dogs with melanoma than in a control population (P ≤ 0.002). Concentrations of VEGF in the serum and plasma of dogs with melanoma were highly correlated (r = 0.867). Ninety‐five per cent of melanoma tissues expressed VEGF. Two staining patterns were detected: diffuse and granular cytoplasmic staining. High blood concentrations of VEGF were correlated to a shorter survival time in dogs receiving definitive therapy (P = 0.002). Survival times were significantly longer in dogs receiving definitive therapy versus palliative therapy (median 496 versus 97 days, P = 0.007). Blood concentrations of VEGF were associated with stage (P < 0.05). Dogs with oral melanoma have increased serum, plasma and tissue concentrations of VEGF. Increased expression of VEGF may be a reasonable target for future therapy of canine oral melanoma.     

Outcomes and prognostic variables associated with primary abdominal visceral soft tissue sarcomas in dogs: A Veterinary Society of Surgical Oncology retrospective study

Primary abdominal visceral soft tissue sarcomas (STSs) are rare tumours in dogs with little information available on outcomes. The goal of this retrospective, multi‐institutional study was to describe the common tumour types, location and prognostic factors associated with primary abdominal visceral STSs. Medical records were searched for dogs with primary abdominal visceral STSs at six institutions and were retrospectively reviewed. Tumours were graded using the previously described grading scheme for STSs of the skin and subcutis when information in the histopathology report contained adequate details. Forty‐two dogs were included in the study. Five dogs had grade I tumours, 11 had grade II and 15 had grade III tumours. The most common tumour type was leiomyosarcoma (38.1%). The most common tumour locations were the spleen (47.6%) and small intestine (23.8%). The local recurrence rate was low (4.7%). Metastasis was present at the time of surgery in 23.8%, and the overall metastatic rate was 40.4%. Mitotic index of ≥9 was associated with significantly shorter survival time (MST 269 days) compared with a mitotic index of <9 (MST not reached). The MST for grade I STSs was not reached, was 589 days for grade II and 158 days for grade III. Dogs with grade III tumours were more likely to develop metastatic disease. Neither location of the primary tumour nor the histologic subtype was associated with survival time. Histologic grading of abdominal visceral STSs using the previously described scheme is prognostic and should be provided on histopathology reports.     

Palliative radiotherapy as treatment for non-resectable soft tissue sarcomas in the dog – a report of 15 cases

Fifteen dogs with various non‐resectable soft tissue sarcomas were treated with a palliative protocol of Cobalt⁶⁰ radiation. Twelve (80%) of the 15 tumours were fibrosarcomas and haemangiopericytomas. Total tumour radiation dose was 24 Gy, given in three 8 Gy fractions, on days 0, 7, 21 or weekly. Thirteen tumours (87%) responded with stable disease; median time to progression and median survival time were 263 and 332 days, respectively. Radiation toxicity was negligible. The survival and local control with this palliative protocol are almost comparable with curative intent primary radiotherapy.     

Basic fibroblast growth factor stimulates epithelial cell growth and epithelial wound healing in canine corneas

Objective  To evaluate the effect of basic fibroblast growth factor (bFGF) on the proliferation of canine corneal epithelial cells and epithelial wound healing.
Animal studied  Canine corneal epithelial cells from the corneas of euthanized dogs and corneal epithelial wounds on one eye from each of 24 dogs.
Procedures  The proliferation of corneal epithelial cells in vitro was measured using the methylthiazolyl-tetrazolium (MTT) assay. A corneal wound on one eye of each dog was made with a corneal trephine (6 mm diameter). Four concentrations of bFGF, 0, 100, 500, and 1000 ng/mL, were applied to the affected eyes of dogs, t.i.d. Fluorescein staining was used to assess closure of the corneal epithelial wound.
Results  The addition of bFGF resulted in a significant increase in epithelial proliferation at 24 h after culture, except 1 ng/mL bFGF. Cells with all bFGF treatments proliferated significantly at 48 and 96 h compared to those in the non-bFGF group. bFGF at a concentration of 10 ng/mL promoted cell proliferation maximally. The wound healing rate in the bFGF-treated groups was greater than that in the control. All corneal wounds in bFGF-treated corneas closed by day 7, whereas two of six corneal wounds in the control showed poor healing. None of the eyes developed corneal clouding or neovascularization during the experiment.
Conclusions  Basic fibroblast growth factor accelerated the proliferation of canine epithelial cells and effectively promoted corneal epithelial wound healing.     

牛碱性成纤维细胞生长因子基因在大肠杆菌中的表达及其表达产物的生物学活性

采用巢式PCR方法克隆了牛18ku-bFGF基因完整的编码序列,并构建了原核表达载体pET-28a-bFGF,将其转化大肠杆菌BL21,在25℃低温条件下,用0.5 mmol/L IPTG诱导表达5 h,用Ni-NTA亲和纯化细胞裂解上清液,经Western-blotting检测,结果显示,在特定的诱导条件下,重组牛bFGF基因在大肠杆菌中获得了表达,并且主要以可溶性状态存在于细胞中。经检测,纯化后的重组蛋白能显著促进成纤维细胞的增殖（P〈0.05）,其活性与商品用重组人18ku-bFGF没有差异（P〉0.05）。表明,所获得的可溶性重组牛18ku-bFGF蛋白具有较高的生物学活性,可用于后续研究工作。     

Serum vascular endothelial growth factor in dogs with soft tissue sarcomas

This work aimed to evaluate serum vascular endothelial growth factor (VEGF) in 25 dogs with soft tissue sarcoma, and in 30 healthy dogs. Blood was collected once time from the control animals and three times, in the same way, from animals with sarcoma. Blood count was performed in the blood collected, and serum VEGF was measured by enzyme‐linked immunosorbent assay quantitative method. Serum VEGF in control animals was similar to patients with soft tissue sarcoma. There was a reduction in serum VEGF after the sarcoma resection. There was positive correlation between serum VEGF and neutrophil counts, and negative between VEGF and hemoglobin content in animals with sarcoma. Animals with hemangiopericytoma showed higher serum VEGF levels compared to the patients with malignant peripheral nerve sheath. Circulating blood cells can contribute to elevate VEGF serum concentrations in dogs with soft tissue sarcomas and a possible role of VEGF in the angiogenesis of these tumors.     

Quantification of epidermal growth factor receptor (EGFR) in canine mammary tumours by ELISA assay: clinical and prognostic implications

The involvement of epidermal growth factor receptor (EGFR) is well established in human breast cancer, however, in canine mammary tumours (CMT), including inflammatory mammary carcinomas (IMC), still needs to be clarified. Enzyme immune assay techniques were used for EGFR determinations in tumour tissue from 45 bitches with CMT and in normal mammary glands from eight control dogs. Higher tissue EGFR levels were found in CMT compared with controls (P < 0.05). In malignant CMT, tissue EGFR elevated concentrations were statistically significantly associated with tumour relapse and/or distant metastasis during follow‐up and with reduced disease‐free and overall survival times. The IMC cases had the highest tissue EGFR levels compared with other malignant non‐IMC tumours (P < 0.001). The results support the hypothesis that EGFR levels influence prognosis in malignant CMT, suggesting that EGFR may represent a therapeutic target in cases of high histological aggressiveness and especially in cases of metastatic phenotype and poor prognosis.     

Expression of fibroblast growth factor 21 in the liver of dairy cows in the transition period and during lactation

Fibroblast growth factor 21 (FGF21) has been identified as a novel hormonal factor involved in the regulation of metabolic adaptations during energy deprivation. The present study aimed to investigate the expression of the FGF21 gene in the liver of dairy cows during the transition from pregnancy to lactation. Therefore, the relative mRNA abundance of FGF21 in liver biopsy samples of 20 dairy cows in late pregnancy (3 weeks pre‐partum) and early lactation (1, 5, 14 weeks post‐partum) was determined. It was observed that hepatic mRNA abundance of FGF21 at 1 week post‐partum was dramatically increased (110‐fold) compared to 3 weeks pre‐partum (p < 0.001). With progress of lactation, mRNA concentration of FGF21 was declining; nevertheless, mRNA abundance at 5 and 14 weeks post‐partum remained 25‐ and 10‐fold increased compared to 3 weeks pre‐partum (p < 0.001). Using a gene array technique, it was found that many genes involved in fatty acid oxidation, gluconeogenesis and ketogenesis were up‐regulated during early lactation compared to late pregnancy. Moreover, there were positive linear correlations between hepatic mRNA concentration of FGF21 and mRNA concentrations of genes involved in ketogenesis as well as carnitine synthesis and carnitine uptake at various time‐points during lactation, indicating that FGF21 could play a role in ketogenesis and carnitine metabolism in the liver of dairy cows (p < 0.05). In overall, the present study shows that expression of the FGF21 gene is strongly up‐regulated during the transition period. It is assumed that the up‐regulation of FGF21 might play an important role in the adaptation of liver metabolism during early lactation in dairy cows such as in other species.     

Regulation of fibroblast growth factor 9 on the differentiation of goat intramuscular adipocytes

It has been found that fibroblast growth factor receptor (FGF-FGFR) signaling can regulate the expression of adipocyte differentiation genes. FGF9 is one of the members of FGFs that mainly binds receptors FGFR2 and FGFR3. FGF9 is highly expressed in the adipose tissue of humans and mice, but there are few reports on the role of FGF9 in goat intramuscular adipocyte differentiation. Therefore, this study explored the effect of FGF9 on adipocyte differentiation through cell culture, interference, and overexpression. The expression of receptors FGFR1–FGFR4 in adipocyte differentiation and their effects on differentiation were detected to screen receptor gene of FGF9. Finally, the interaction between FGF9 and the receptor was tested by cotransfection. Our results showed that FGF9 interacts with FGFR2 to inhibit goat intramuscular adipocyte differentiation by regulating peroxisome proliferator-activated receptor gamma (PPARγ) and preadipocyte factor 1 (Pref1), which is a data support for subsequent pathway research.     

Expression of vascular endothelial growth factor receptor-1 and -2 in normal and diseased canine eyes

Objective To immunohistochemically evaluate expression of vascular endothelial growth factor receptor‐1 (VEGFR1) and ‐2 (VEGFR2) in ocular tissue of healthy dogs and dogs affected with primary glaucoma, uveitic glaucoma, and intraocular neoplasia. Sample population Enucleated globes from five dogs with primary glaucoma, five dogs with uveitic glaucoma, six dogs with intraocular neoplasms and three ophthalmically normal control dogs. Procedure Ocular tissues were obtained from enucleated globes of clinical cases or immediately following euthanasia for control dogs. Tissue sections were stained immunohistochemically for VEGFR1 and VEGFR2 via standard techniques and vascular tissue was qualitatively evaluated. Vascular endothelial VEGFR1 and VEGFR2 expression patterns are reported for normal and diseased ocular tissues. In addition, VEGFR1 and VEGFR2 expression patterns are reported for all normal ocular tissues. Results A constitutive expression pattern was detected for VEGFR1 by ocular vascular endothelial cells as well as nonvascular cells in the cornea, uvea, lens, and retina. VEGFR2 demonstrated limited expression in normal ocular tissue, but was widely expressed in vascular endothelium of diseased eyes, particularly in pre‐iridal fibrovascular membranes. Conclusions The results of this study suggest a role for VEGF receptors in both physiologic and pathologic angiogenesis in canine ocular tissue. Manipulation of this pathway may be a rational consideration for therapeutic intervention in canine ocular disease exhibiting pathologic neovascularization.     

Soft tissue sarcomas in the pharyngeal region of a 5‐year‐old Quarter Horse mare

This case report describes the identification of multiple soft tissue sarcomas in the pharyngeal region of a 5‐year‐old Quarter Horse mare. Diagnostic work‐up included physical examination, radiography, ultrasonography, endoscopic examination of upper airways and guttural pouch, and post mortem examination with histopathology. Humane euthanasia was indicated due to the chronicity of the condition, prognosis and financial constraints.