Effect of anaesthesia on cell-mediated immunity in dogs undergoing mastectomy for mammary cancer.

OBJECTIVE: To investigate if anaesthesia for canine cancer mastectomy further influences host cell-mediated immunity (CMI) promoting cancer progression. STUDY DESIGN: A randomized, controlled, blinded clinical study. ANIMALS: A total of 20 bitches with malignant mammary tumours of clinical stage II or III undergoing the same type of mastectomy (regional mastectomy). METHODS: Dogs were randomly allocated to one of two anaesthetic groups (10 per group). The anaesthetic protocol of group A used minimally immunosuppressive drugs (tramadol, robenacoxib, propofol), whereas that of group B (control) used more immunosuppressive drugs (morphine, fentanyl, thiopental, isoflurane). For each animal, measurements of white blood cells (WBCs), neutrophils and lymphocytes, and flow cytometric assessment of T cells (CD3+), helper T cells (CD4+), cytotoxic T cells (CD8+) and CD5low+ T cells were performed prior to anaesthesia (day 0) and on days 3 and 10 postsurgery. Data were analysed using a General Linear Model for repeated measures and presented as mean ± standard deviation, p ≤ 0.05. RESULTS: In all animals, on day 3, WBCs and neutrophils were significantly increased (p < 0.0005), while flow cytometry revealed significantly decreased relative percentages of T cells (CD3+) (p = 0.003) and their subpopulations CD4+ (p = 0.006), CD8+ (p = 0.029) and CD5low+ (p = 0.031). Specifically, on day 3, the cytotoxic T cells (CD8+) were significantly decreased (p = 0.05) only in group B, whereas the CD4+ (p = 0.006) and CD5low+ (p = 0.008) T cells in group A. The only significant difference between groups was found preoperatively in the CD4+/CD8+ ratio, which was higher in group A (p = 0.006). CONCLUSIONS AND CLINICAL RELEVANCE: In dogs with mammary cancer undergoing regional mastectomy, a significant decrease in components of CMI was observed on day 3 postsurgery in both anaesthetic groups. Some indication, however, for better preserved cellular immunity by less immunosuppressive anaesthetic/analgesic drugs was detected, rendering their use advisable.

Adipose-derived stromal vascular fraction aids epithelialisation and angiogenesis in an animal model.

OBJECTIVE: Limited data exist regarding the correlation between adipose-derived stromal vascular fraction (SVF) and wound healing. The aim of this study was to investigate the direct effect of intradermally injected SVF on full-thickness cutaneous wounds in a murine model. METHOD: Wistar rats were divided into three groups (A, B and C) according to their day of euthanasia (day 7, 16 and 21). Inguinal fat pad was excised and SVF enzymatically extracted. Full-thickness cutaneous wounds were created on each side of the dorsum; SVF injected intradermally at one side while the contralateral wound served as control receiving normal saline. Postoperatively, evaluation of wound healing was performed by planimetry (percentages of wound contraction, epithelialisation and total wound healing) on days 0, 3, 5, 7, 10, 13, 16 and 21, and histology and immunochemistry (cellular infiltration score, collagen production score, neoangiogenesis and epithelial thickness) on days 7, 16 and 21. RESULTS: Despite the high rate of wound contraction, it was significantly lower in the SVF-treated wounds on day 21 (p=0.037). On days 13, 16 and 21, the percentages of epithelialisation were higher in the SVF-treated wounds compared with control wounds (p=0.026, p=0.048 and p=0.05, respectively). Histologically, the number of new vessels was significantly higher in the SVF-treated wounds compared with controls on days seven (p=0.028) and 16 (p=0.027). This was also confirmed by immunohistochemistry. No significant differences were found between treated and control wounds regarding cellular infiltration score, collagen production score and epithelial thickness. CONCLUSION: Data indicate that intradermally injected SVF increases angiogenesis and enhances epithelialisation in full-thickness cutaneous wounds in rats.

ASSESSMENT OF SENTINEL LYMPH NODE METASTASIS IN CANINE MAMMARY GLAND TUMORS USING COMPUTED TOMOGRAPHIC INDIRECT LYMPHOGRAPHY.

In the staging process of the breast cancer, demonstrating metastasis of the sentinel lymph node (SLN) has an important prognostic value, in both humans and animals. The aim of this prospective case-control study was to determine the diagnostic value of computed tomographic indirect lymphography (CT-LG) for detecting SLN metastasis in dogs with mammary cancer. Thirty-three female dogs with tumors in the abdominal and inguinal mammary glands were prospectively selected and subjected to CT-LG, 1 and 5 min after injection of 1 ml of contrast agent (iopamidol) in the subareolar tissue of the neoplastic and the contralateral normal mammary glands. The pattern of postcontrast opacification, degree of postcontrast enhancement, and size and shape were assessed in 65 SLNs in total and were correlated with histopathological findings. The absence of opacification or heterogeneous opacification 1 min after contrast medium injection showed the highest sensitivity, specificity, and accuracy (93%, 100%, and 98.4%, respectively). In images taken 1 min after injection, an absolute density value lower than 444 Hounsfield units (HU) in the center of the SLN also provided significant sensitivity and specificity (93.8% and 75%, respectively). The size and shape of the SLN (maximum and minimum diameter, maximum/minimum diameter ratio, maximum diameter/height of fifth thoracic vertebral body ratio) showed the lowest sensitivity and specificity. Results of this study support the hypothesis that CT-LG could help in the assessment of SLN metastasis in cases of mammary gland tumors in dogs.

Evaluation of Survival of Subdermal Plexus Skin Flaps in 8 Cats After Injection of Platelet Rich Plasma.

The aim of the present study was to evaluate the effect of platelet-rich plasma injection on the survival of experimentally developed subdermal plexus skin flaps in cats. Two flaps, 2 cm wide and 6 cm long were created bilaterally in the dorsal midline in 8 cats. Each flap was randomized to 1 of 2 groups: platelet-rich plasma injection and control. After flap development, the flaps were immediately placed back on the recipient bed. Then 1.8 mL of platelet-rich plasma was equally injected into 6 different parts of the treatment flap. All flaps were evaluated macroscopically daily and on days 0, 7, 14, and 25 by planimetry, Laser Doppler flowmetry, and histology. Mean flap survival on day 14 was 80.437 % (±22.745) for the treatment group and 66.516 % (±24,12) for the control group with no statistically significant difference between the 2 groups (P = .158). Histologically, a significant difference was found between the base of the PRP and the control flap in edema score on day 25 (P = .034). In conclusion, there is no evidence to support the use of platelet-rich plasma in subdermal plexus flaps in cats. However, the use of platelet-rich plasma may aid in reducing edema of subdermal plexus flaps.

Effect of major versus minor mastectomy on host immunity in canine mammary cancer.

Surgical procedures can affect host immunity proportionally to the extent of surgical trauma. In cancer cases, surgery-induced immunosuppression can potentially promote tumour metastasis. The aim of this study was to investigate, in bitches with malignant mammary tumours, whether major surgery (total unilateral mastectomy or bilateral regional mastectomy) has a more negative effect than minor surgery (unilateral regional mastectomy) on components of host immunity. Twenty bitches with mammary cancer of clinical stage II or III were allocated to group A (minor surgery) or group B (major surgery) of 10 animals each receiving the same anaesthetic protocol for mastectomy. Immune cell measurements in blood [number of leukocytes, neutrophils, lymphocytes and platelets, and relative percentages of T-lymphocytes (CD3+) and their CD4+, CD8+ and CD5low+ subpopulations] were performed before anaesthesia (day 0) and on days 3 and 10 post-mastectomy. On day 3, leukocytes, neutrophils and platelets numbers were higher (p = 0.016, 0.032 and 0.017, respectively) in group B than in group A. For all 20 bitches, T-lymphocytes and the CD4+, CD5low+ T-cells were significantly decreased on day 3, but no significant differences were noted between groups. Minor mastectomy seemed to preserve innate immunity better than major mastectomy, but cellular immunity was rather equally affected.

Locally Injected Autologous Platelet-Rich Plasma Improves Cutaneous Wound Healing in Cats.

Cutaneous defects in cats are commonly encountered in clinical practice, and healing can be accomplished by first or second intention. Platelet-rich plasma (PRP) is characterized by a plasma concentration containing a large number of platelets in a small volume of plasma. The objective of the present study was to record the efficacy of PRP infiltration in open wounds in laboratory cats. Six wounds were created in the dorsal midline of eight laboratory cats, with the wounds of one side designated as the PRP group and the wounds of the other side as the control group. Wound healing was evaluated by daily clinical examination, planimetry, laser Doppler flowmetry, and histologic examination on days 0, 7, 14, and 25, and by measurement of metalloproteinases (MMPs)-2 and -9 and tissue inhibitor metalloproteinase (TIMP)-1 on days 0, 14, and 25. Based on the results of the present study, the mean time for full coverage with granulation tissue was shorter in the PRP group, the mean contraction and total wound healing percentage were increased compared to the control group, and finally, the perfusion measured with laser Doppler flowmetry was higher in the PRP group during all examination days. In conclusion, this is the first study focusing on the topical application of PRP in the treatment of open wounds in laboratory cats, and our results are encouraging-showing a more rapid healing in the PRP group.

Evaluation of blood T-lymphocyte subpopulations involved in host cellular immunity in dogs with mammary cancer.

Cancer-bearing patients are often immunosuppressed. In dogs with mammary or other cancers, various alterations in blood cell populations involved in host cellular immunity have been reported; among these cell populations some T-lymphocyte subsets play an important role against cancer. The purpose of the present study was to investigate any alterations in circulating T-lymphocyte subpopulations involved in cellular immunity in bitches with mammary cancer, in comparison to age-matched healthy intact bitches. Twenty eight dogs with mammary cancer and 14 control dogs were included in this study. Twelve out of the 28 bitches had mammary cancer of clinical stage II and 16/28 of stage III. Histological examination revealed that 23/28 animals had carcinomas, 3/28 sarcomas and 2/28 carcinosarcomas. White blood cell, neutrophil and lymphocyte absolute numbers were measured by complete blood count. Furthermore, blood T-lymphocyte population (CD3+) and the subpopulations CD4+, CD8+ and CD5low+ were assessed by flow cytometry. White blood cell and neutrophil but not lymphocyte absolute numbers were higher (P=0.003 and P=0.001, respectively) in cancer patients than controls. Flow cytometric analysis revealed that the relative percentage of T-lymphocytes (CD3+) and of CD4+, CD8+ subpopulations was lower (the CD4+/CD8+ ratio was higher), whereas the percentage of CD5low+ T-cells was higher, in dogs with cancer compared to controls; however, a statistically significant difference was found only in the case of CD8+ T-cells (P=0.014), whereas in the case of the CD4+/CD8+ ratio the difference almost reached statistical significance (P=0.059). Based on these findings, it can be suggested that, although the absolute number of blood lymphocytes is unchanged, the relative percentages of T-lymphocyte subpopulations involved in host cell-mediated immunity are altered, but only cytotoxic CD8+ T-cells are significantly suppressed, in dogs with mammary cancer of clinical stage II or III compared to age-matched healthy controls.

Evaluation of intra-articular injection of autologous platelet lysate (PL) in horses with osteoarthritis of the distal interphalangeal joint.

BACKGROUND: Regenerative medicine has become one of the most promising therapies of equine osteoarthritis. Platelet lysate (PL) is rich in bioactive proteins and growth factors that play a crucial role in tissue healing. OBJECTIVE: To evaluate the efficacy of intra-articularly injected autologous PL in equine athletes with naturally occurring osteoarthritis. ANIMALS AND METHODS: Fifteen warmblood geldings aged 8-19 years with osteoarthritis of the distal interphalangeal joint were included in this study. They were randomly divided into two groups; 10 horses received intra-articular injections of PL and 5 of normal saline (controls). Before treatment, platelet-derived growth factor (PDGF) levels in basal plasma and prepared PL were estimated. Each joint was injected twice within a three-week period. Lameness was evaluated using the American Association of Equine Practitioners grading system, before treatment and 10 days after each intra-articular injection. Horses were examined fortnightly for one year. Radiographic examination was performed six months post-treatment. The generalized estimating equation test was used for statistical analysis. RESULTS: Acceptable levels of PDGF were detected in PLs (mean ± SD: 258.0 ± 52.3 pg/ml). The majority of horses (9/10) responded positively to PL treatment presenting lower lameness grades (p < 0.0005) compared to controls 10 days after the second injection, and returned to normal athletic activity. Radiographs revealed no changes in osteoarthritis lesions six months after treatment. One year post-injections, however, all horses relapsed to their initial degree of lameness. CONCLUSION: Intra-articularly injected autologous PL is an efficient method for temporarily managing osteoarthritis of the distal interphalangeal joint in athletic horses.

Effects of two occlusive, hydrocolloid dressings on healing of full-thickness skin wounds in cats.

OBJECTIVES: To determine the effects of two occlusive, hydrocolloid dressings on second intention wound healing in cats. METHODS: Three 2×2 cm full-thickness skin wounds were created on each side of the trunk of 10 cats. Two bilateral wounds were bandaged using different hydrocolloid dressings, namely Hydrocoll and DuoDerm while a semi-occlusive pad (Melolin) was applied to the third bilateral wound (control group). Wound planimetry, subjective evaluation of wound healing, and qualitative bacterial cultures were performed on the right-sided wounds, whereas left-sided wounds were subjected to histological examination. RESULTS: Subjective evaluation revealed accelerated (p <0.01) granulation tissue formation in hydrocolloid-treated wounds. The amount of wound exudate was initially greater (p <0.01) in these wounds. Seventeen out of 120 evaluated wounds (14.2%) produced purulent exudate, 11 of which showed Staphylococcus aureus growth. Fewer control wounds (3/17) emitted purulent exudate, but all of them yielded moderate bacterial growth as compared to hydrocolloid-treated wounds (8/17) which had a low incidence of bacterial growth. The only significant difference in histological variables was increased cellular infiltration noted on day 7 in Hydrocoll-treated wounds compared to controls. No significant differences in planimetry variables were found between groups. CLINICAL SIGNIFICANCE: Despite the initially increased amount of wound exudate and the low growth of Staphylococcus aureus, hydrocolloid dressings accelerate granulation tissue formation and can hence be suggested for second intention wound healing in cats.

Markers of lipid peroxidation and α-tocopherol levels in the blood and neoplastic tissue of dogs with malignant mammary gland tumors.

BACKGROUND: Excess reactive oxygen species due to oxidative stress and the ensuing lipid peroxidation are believed to be involved in mammary gland tumor (MGT) pathogenesis. OBJECTIVE: The purpose of this study was to evaluate the extent of lipid peroxidation as evidenced by thiobarbituric acid-reactive substances (TBARS) formation, and the concentration of α-tocopherol as an inhibitor of lipid peroxidation, in blood and neoplastic tissue of dogs with malignant MGT. The correlation between inflammatory cell infiltration score and TBARS or α-tocopherol in MGT was also evaluated. METHODS: Sixteen intact female dogs with malignant MGT and 12 clinically healthy and age/weight-matched controls were included in the study. In all dogs, serum TBARS, α-tocopherol, total cholesterol, and triglycerides were measured. Tissue TBARS and α-tocopherol levels were determined in 1 cm(3) sized tissue samples collected from MGT and adjacent, ipsilateral, normal mammary gland tissue from the 16 affected dogs. The degree of inflammatory cell tumor infiltration was evaluated histologically. RESULTS: There were no statistically significant differences in serum levels of TBARS, α-tocopherol, total cholesterol, and triglycerides between dogs with and without malignant MGT. TBARS were significantly higher, whereas α-tocopherol was lower in neoplastic tissue when compared with normal mammary gland tissue. There was no correlation between TBARS or α-tocopherol concentration and the inflammatory cell infiltration score in neoplastic tissue. CONCLUSION: The increased level of TBARS suggests oxidative stress induction in canine malignant MGT. The origin of this phenomenon is not clear, as a potential oxidative burst could not be attributed to inflammatory cells infiltrating the tumors.

Evaluation of the effectiveness of an ointment based on Alkannins/Shikonins on second intention wound healing in the dog.

The enantiomeric naphthoquinones alkannins and shikonins (A/S) have been established as potent wound healing agents. The purpose of this study was to evaluate the effectiveness of an A/S based ointment for humans on second intention wound healing in the dog, as compared to wound flushing with Lactated Ringer's solution (LRS). Ten mixed breed dogs, aged 2 to 5 y, were used. One 2.5 × 2.5 cm full-thickness skin defect was created on the lateral aspect of each arm for subjective evaluation, laser-Doppler flowmetry (LDF), and planimetry. Additionally, 3 matching 2 × 2 cm wounds were created on opposite sides of the dorsal midline for histologic evaluation. Wounds were treated once daily with the A/S based ointment on the right side and by flushing with LRS on the left until healed (about 20 d). During the healing process, tissue perfusion (mean LDF value) was found to be significantly higher on the side treated with the A/S based ointment compared with the LRS-treated side. Histologically, angiogenesis (on days 4 and 11), collagen production score (on days 4, 11, and 20), and epithelial thickness score (on day 11) were significantly higher in the wounds treated with the A/S based ointment. Wound size, as evaluated by planimetry, decreased significantly from day 0 to day 20 on both sides, but no significant differences were found between the A/S based ointment and LRS-treated wounds.

Naturally occurring isohexenylnaphthazarins and wound healing: experimental study in dogs.

BACKGROUND: The healing efficacy of isohexenylnaphthazarins (IHN) has been well proved on chronic or contaminated wounds. OBJECTIVE: To evaluate the wound healing activity of an experimental ointment containing IHN on acute and noncontaminated wounds in dogs. METHODS: In each of six beagle dogs, four full-thickness skin defects were created bilaterally: one 2 x 2 cm defect on the lateral aspect of each arm for subjective evaluation, laser Doppler flowmetry (LDF), and planimetry and three 1.5 x 1.5 cm defects on opposite sides of the dorsal midline for histologic evaluation. Wounds on the left were treated with an ointment based on IHN and on the right with another based on petroleum jelly (Vaseline) and beeswax. RESULTS: Wound size decreased significantly in both sides. The significantly increased percentage of epithelialization was higher (p = .0274) in the petroleum jelly-treated wounds on day 20. Tissue perfusion (LDF) increased significantly bilaterally in the center of the wound but only in the IHN-treated side cranial to the wound. Histologically, angiogenesis was significantly higher (p = .0431) on day 5 in the IHN-treated wounds compared with the petroleum jelly-treated wounds. Collagen production increased significantly bilaterally. CONCLUSION: The IHN-based ointment promoted some of the proliferative processes, but it did not enhance the overall wound healing of acute, surgically created wounds in dogs.