Efficacy of 5-aminolevulinic acid-based photodynamic therapy in different subtypes of canine mammary gland cancer cells.

Canine mammary gland tumors (CMGTs) are heterogeneous disease and subclassified [sarcomas (S), carcinomas (C), and carcinosarcomas (CS)] according to histopathological differentiation. Photodynamic therapy (PDT) is a promising treatment strategy based on the use of a photosensitizer (PS) activated by light. However, the therapeutic potential of PDT in the treatment of CMGTs has not been investigated, yet. Therefore, the aim of this study was to determine the in vitro protocol of 5-ALA-based-PDT for the treatment of three subtypes of CMGTs, for the first time. The intracellular PpIX florescence intensity was measured for 5-ALA (0.5 and 1 mM). After irradiation with different light doses (6, 9, 12, 18, and 24 J/cm2) for two different modes [continuous wave (CW) and pulse radiation (PR)], the cytotoxic effects of 5-ALA (0.5 and 1 mM) on the subtypes (C, S, and CS) of CMGTs were analyzed by WST-1. Finally, the optimal PDT treatment protocol was validated through Annexin V and AO/EtBr staining. Our results showed that 1 mM 5-ALA for 4-h incubation was the best treatment condition in all subtypes of CMGTs due to higher intracellular PpIX level. After irradiation with different light doses, PR mode was more effective in S primary cells at 9 J/cm2. However, a significant decrease in the viability of C and CS cells was detected at 12 /cm2 in CW mode (p < 0.05). Additionally, 1 mM 5-ALA induced apoptotic cell death in each subtype of CMGTs. Our preliminary findings suggest that (i) each subtype of CMGTs differentially responds to PDT and (ii) the light dose and mode could play an important role in the effective PDT treatment. However, further studies are needed to investigate the role of the different light sources and PDT-based apoptotic cell death in CMGTs cells.

Amounts of tissue magnesium and some trace elements in cats with mammary tumors related to various clinicopathological parameters.

BACKGROUND: Mammary tumors are one of the major malignancies seen in cats. Researchers have indicated the similarity between the epidemiological and clinicopathological patterns of feline mammary tumors and human breast cancer (HBC). In recent years, the investigation of trace elements in cancer tissues becomes prevalent in HBC due to the role of these elements in biochemical and physiological processes. This study, it is aimed to evaluate some trace elements in feline mammary tumors according to clinical and pathological findings. METHODS: A total of 60 tumoral masses from 16 female cats with mammary tumors were included in the study. The study groups were formed according to histopathology as malignant epithelial tumor (MET; n = 39) and hyperplasia and dysplasia (H&D; n = 21). Copper (Cu), Iron (Fe), Magnesium (Mg), Manganese (Mn), Selenium (Se) and Zinc (Zn) trace elements in mammary tissues were analyzed by using an inductively coupled plasma-optical emission spectrophotometer. RESULTS: The mean age and weight of the cats were 11.75 ± 0.75 years and 3.35 ± 0.21 kg; respectively. Eleven of 16 cats were intact whereas the rest of them had been spayed. Metastases were observed in 10 cats. Tissue Mg level in group MET was significantly higher than in group H&D (P < 0.01) while the other elements had not significant differences between the groups. In group MET, analyzed elements were not statistically significant related to the inflammation, ulceration and invasion to the peripheral muscle (P > 0.05). However, tissue Fe level was significantly higher in T2 than in T3 (P < 0.05). The mean levels of tissue Fe, Mg and Mn had significant differences related to histological grading as P < 0.01, P < 0.05 and P < 0.001; respectively. A mild to severe correlation was found between tissue Zn and Se, Cu, Fe, Mg, and Mn levels. CONCLUSION: Tissue Mg and some trace elements were evaluated in feline mammary tumours in regard to various clinicopathological parameters. Tissue Mg level was sufficient to differentiate the malignant epithelial tumors from hyperplasia and dysplasia. However, Mn and Se tended to distinguish different tumor types. Tissue Fe, Mg and Mn had significant differences related to histological grading. Also, the Fe level was significantly higher in T2 than in T3 and Zn level tended to be higher in T3 than in T1. It was concluded that Mg, Se, Mn, Fe, Cu and Zn provided useful information on the pathogenesis of feline mammary tumors. Further research is needed on the tissue and serum concentrations of trace elements which may provide valuable information for the disease prognosis.

Evaluation of vascularization in the walls of preovulatory follicles in mares with endometritis.

Endometritis is a major cause of infertility in mares. The aim of this study is to evaluate the preovulatory follicle (POF) vascularization (A mix, A red, A blue), POF diameter, POF wall thickness, and uterine diameters in mares with or without endometritis. Ten healthy mares and 10 mares with endometritis diagnosed by the combination of transrectal palpation, ultrasonographic examination, and cytology brush were enrolled in the study. Data of the groups obtained at 2 days before the ovulation (day -2) were compared with Student's t-test. Correlations of the parameters were determined by the Pearson correlation test. To the best of our knowledge, this is the first report indicating a lower vascularization of A mix, A red, A blue, and a significantly lower wall thickness in the POFs of the mares with endometritis compared to the healthy ones (p < 0.01, p < 0.05, p = 0.06, and p < 0.001, respectively). Also, a striking and novel inverse correlation between POF wall thickness and uterine diameter (r = -0.785, p < 0.001) and moderate correlations between POF wall thickness and POF vascularizations of A mix and A red (r = 0.436, p = 0.055, and r = 0.427, p = 0.060, respectively) were determined.

Evaluation of changes in Doppler ultrasonography indices and levels of maternal serum angiogenic factors throughout pregnancy in ewes.

We evaluated the changes in the pulsatility index (PI) and resistance index (RI) of fetal and maternal vessels and maternal serum angiogenic factors throughout pregnancy in 20 Kivircik ewes. They were examined for pregnancy detection on Day 30 after mating. The PI and RI during the pregnancies were followed using Doppler ultrasonography (USG) of the uterine artery, umbilical artery, fetal aorta, and fetal vena cava. Doppler USG was performed every 15 days beginning from the 40th day after mating. Blood samples were collected every 15 days starting from the day of mating. Nitric oxide (NO), vascular endothelial growth factor, basic fibroblast growth factor, and angiopoietin-1 and -2 levels were measured in maternal serum. A gradual decline was observed in the PI and RI of the umbilical artery beginning from Day 75 of pregnancy. Similarly, the RI of the uterine artery decreased on Day 135. Embryonic resorption was detected in two ewes with an increased PI and RI in the uterine artery. A significant and gradual decrease in the maternal serum vascular endothelial growth factor level was observed throughout pregnancy. The maternal serum NO level increased beginning from Day 135 of pregnancy. The results of this study illustrate the progressive changes in the Doppler USG findings of fetomaternal vessels and maternal serum angiogenic factors that occur throughout ewe pregnancy. The umbilical and uterine artery Doppler USG findings and maternal serum NO concentration may be important parameters for evaluation of the course of pregnancy in ewes. The results of this study should be compared with those of further studies that include compromised pregnancies and nonpregnant ewes.

Evaluation of platelet indices and complete blood count in canine mammary tumors

Malignant mammary tumors in humans and bitches cause hematological disorders such as anemia, erythrocytosis, thrombocytosis, hyperproteinemia, and leucopenia. Novel studies have been conducted on the predictive and prognostic values of platelet (PLT) indices in human breast cancer (HBC). However, there is little information about the alterations in hematological parameters in canine mammary tumors (CMTs). The aims of this study were to evaluate the platelet indices and complete blood count (CBC) parameters in bitches with and without mammary tumor and to assess the above mentioned parameters with regard to histological tumor types and grades. A total of 71 bitches were enrolled in this study. The bitches in the study group were divided into 2 groups which consisted of malignant epithelial mammary tumors (group EMT; n = 43) and malignant mixed mammary tumors (group MMT; n = 12). Control group (group C) consisted of clinically and gynaecologically healthy 16 bitches. Blood samples were obtained to perform the CBC and PLT indices analysis. Histopathological examinations were carried out under a light microscope. Histological tumor types and malignancy grades were classified. The bitches with mammary tumor showed significantly increased PLT values and decreased hematocrit (HCT), hemoglobin (HGB) and mean corpuscular hemoglobin (MCH) values versus the healthy ones, regardless of the tumor type. However, in comparisons with the group C, mean platelet volume (MPV) and mean corpuscular hemoglobin concentration (MCHC) values were different only in the group MMT, while plateletcrit (PCT) and mean corpuscular volume (MCV) values were different only in the group EMT. Also white blood cell (WBC), PLT and PCT values were higher than the referenced laboratory ranges in grade 3 tumors. In the presented study, MPV was considerably correlated with PLT, platelet distribution width (PDW) and PCT. Also, PCT and PLT had high sensitivity and specificity to distinct EMT and MMT from the healthy bitches. Microcytic and hypochromic anemia occurs due to the decrease in the amount of HGB. Levels of MCV, MCH, and MCHC in the HBC group were reported to be significantly lower than in humans without breast cancer. Although anemia did not occur in EMT and MMT groups, obtained significances in the HCT, HGB, MCV, MCH, and MCHC levels between the bitches with and without mammary tumor were in line with the previous reports. In this study, WBC levels in grade 3 tumors were significantly higher than grade1 tumors (P < 0.05). Whereas levels of WBC in grade 1 and grade 2 tumors were in referenced laboratory ranges, it was higher in grade 3. Increased level of WBC in grade 3 was supposed to be due to the rise in malignancy as previously reported. Thrombocytosis was detected in 48.83% and 41.66% of the bitches in EMT and MMT groups, respectively. The higher percentage of CMTs with thrombocytosis in this study might be due to the difference in referenced upper limit of PLT in previous studies. The elapsed time between tumor formation and clinical presentation could be another influencing factor. Although PLT and PCT values were not significant according to the histological grading in this study, both parameters were found to be higher in grade 3 than the normal reference values. Further studies conducted with higher populations may lead the differences in these parameters to significance. With the support of further studies, alterations in the above mentioned parameters in bitches may contribute in the diagnosis process, management of treatment and may constitute an easy way to have an idea about the prognosis of mammary tumors.(AU)

Osteoporotic risk and physeal closure in prepubertal ovariohysterectomized cats.

We aimed to examine the early effects of prepubertal ovariohysterectomy (P-OHE) on bone loss and proximal physeal closure in cats. Fourteen kittens randomly underwent P-OHE or sham operations (S-OP) at three months (mo) of age and were allocated to group I and group II. Each mo between four and nine mo of age, dual-energy X-ray absorptiometry (DEXA) scans were performed to determine the total body bone mineral density (BMD) and bone mineral content (BMC). Proximal radial physeal closure and radial length were determined by radiography. Bone-specific alkaline phosphatase (BAP), carboxy-terminal collagen teleopeptide (CTX), 17-ß estradiol, progesterone, calcium (Ca) and phosphorus (P) were measured in the serum samples. No significant differences were observed between the groups in terms of BMD, BMC, BAP, BAP/CTX, P, progesterone and body weight (BW) (between 4 and 9mo) and for Ca (between 5 and 9mo) and for CTX levels (between 4 and 8mo). The 17-ß estradiol was significantly higher at 6, 8 and 9mo of age in the S-OP group due to puberty (P=0.02, P=0.03 and P=0.02 respectively). Although there was a significant difference (P=0.0002) between the P-OHE and S-OP groups in terms of the proximal radial physeal closure times (7.43±0.20mo and 6.14±0.14mo, respectively), no significant difference was observed for the mean radius length (10.59±0.10cm and 10.06±0.27cm, respectively) at the last evaluation time. In conclusion, prepubertal ovariohysterectomized cats do not have any osteoporotic risks until nine mo of age and exhibit a delayed physeal closure time without a change in radius length.

Is uterine blood flow influenced by hCG and mare age?

Doppler ultrasonography is a noninvasive technique which enables us to follow the physiologic and physiopathologic changes in blood flow in tissues. It is becoming an essential tool in veterinary medicine, especially in theriogenology. Twenty-seven Arabian mares were grouped by age ('young', 3-10 y, n=15; 'old', 19-23 y, n=12). The uterine arteries of the mares were examined using Doppler ultrasonography when an ovarian follicle ≥35mm was visible (Day -1). After these measurements, human chorionic gonadotropin (hCG; 2500IU) was administered to 14 mares selected randomly. One day later (Day 0) Doppler ultrasonography was repeated and then the dominant follicles were aspirated to collect follicular fluid in all groups. On the next day (Day +1), Doppler indices of the uterine artery blood flow were measured again. Blood samples were also collected just prior to ultrasonography, for measuring serum estradiol and progesterone levels. We found that preovulatory hCG administration had no significant effects on uterine artery blood flow indices, or serum or follicular fluid estradiol concentrations. The uterine artery resistance index might decrease in young mares after ovulation, possibly because of increased uterine perfusion.

Lambing rates and litter size following carazolol administration prior to insemination in Kivircik ewes.

The effect of carazolol on the ease of penetrating the cervix during artificial insemination, lambing rate and litter size was studied using 1.5-4.0-year old Kivircik ewes in an incomplete 3 x 2 x 2 experimental design. All of the ewes in this study were synchronized for oestrus by insertion of a progesterone impregnated vaginal sponge for 12 days and administration of 400 IU PMSG at sponge withdrawal. Three methods of service were compared: natural service, artificial insemination (AI) with fresh semen, or AI with frozen semen. Two times of insemination (fixed time AI versus AI at observed oestrus) were compared on the fresh and frozen AI treatments. The absence (control) or use of carazolol (carazolol; 0.5mg/ewe i.m. 30 min before mating) was the third factor in the design and penetration of the cervix by the insemination pipette was assessed as shallow (<10mm), middle (10-20mm) or deep (>20mm). Natural service ewes were only mated at observed oestrus. Consequently, the factorial design was incomplete and there were a total of 10 treatments each represented by 30 ewes. Natural service resulted in a significantly (P<0.05) higher lambing rate and litter size (86%; 2.0+/-0.05 lambs/ewe) than AI using fresh (65%; 1.6+/-0.1 lambs/ewe) or frozen (40%; 1.4+/-0.14 lambs/ewe) semen. For AI animals the lambing rate and litter size were not significantly different when service was at a fixed time (50%; 1.5+/-0.12 lambs/ewe) or at observed oestrus (56%; 1.5+/-0.12 lambs/ewe). Carazolol did not permit complete cervical penetration in any ewe. Deep penetration of the cervix at AI was achieved in 33% of untreated (control) and 48% of carazolol treated ewes (P<0.05). However, the proportion of ewes in which penetration of the cervix and semen deposition was greater than shallow was similar for control (82%) and carazolol (85%), and lambing rate and litter size were similar for both treatments. Over the three service methods, the lambing rate was 56% for control and 63% for carazolol (NS) and litter size was similar for both treatments. It was concluded that the carazolol treatment used prior to natural mating or AI in this experiment did not improve lambing rate or litter size in Kivircik ewes.