Veterinary Research Forum

Abstract A 4.5-year-old, nulliparous female New Zealand White Rabbit (Oryctolagus cuniculus) was presented for evaluation of a mammary mass located in right axillary mammary gland. The surgically excised mammary mass was submitted for histopathological and immunohistochemical evaluation. Histopathologically, neoplastic cells were arranged in sheets and nests of irregular sizes. The tissue was composed of amorphous eosinophilic material and necrotic cells. The neoplastic cells had round to oval nuclei with prominent nucleoli, scant basophilic cytoplasm and high mitotic activity. Cytokeratin and vimentin expressions of neoplastic cells were observed in immunohistochemical findings. Based on the microscopic characteristics and immunohisto-chemical staining, a diagnosis of a non-invasive comedocarcinoma was established. In this case, cytokeratin positivity confirmed the epithelial origin, whereas the vimentin positivity supported the presence of epithelial-mesenchymal transition and/or increased malignancy.

Abstract Significant advancements in imaging and surgical methodologies have led to more frequent performance of neurosurgical procedures such as laminectomy in both animal and human patients. Epidural fibrosis (EF) is defined as the excessive formation of scar tissue in the epidural space after lumbar laminectomy, often resulting in recurring postoperative pain. Given the association between postoperative hematoma accumulation at the laminectomy site and the development of EF, the present study aimed to evaluate the preventive impact of tranexamic acid (TXA), an antifibrinolytic agent with well-recognized hemostatic properties across various surgical fields. A rabbit laminectomy model was constructed to assess its effectiveness in reducing EF formation. A total number of 18 adult New Zealand White male rabbits were randomly divided into two groups: The control (saline) group and the treatment (topical TXA) group. Each rabbit underwent a two-level laminectomy at L3-L4. The treatment group received 5.00 mL of 100 mg mL^-1 TXA solution applied topically to the laminectomy site, while the control group received 5.00 mL of saline. Postoperative evaluations included magnetic resonance imaging at week six to assess EF, followed by histopathological examinations to evaluate fibroblast cell density in scar tissue, EF grading and thickness of the dura mater. The analysis of magnetic resonance imaging and histopathologic data revealed significant differences between the two groups indicating that topical administration of TXA might be a promising approach for preventing EF.

Abstract Cryopreservation and re-transplantation of ovarian tissue is a relatively new technique to preserve fertility. This study evaluated the preservation of ovarian follicles after the vitrification-thawing procedure by implanting ovarian fragments into the gluteus muscle of rabbit. Bilateral ovariectomy was performed on each rabbit. The cortices were separated from the medullae of ovaries and divided into four fragments. One fresh cortical fragment was used as a control, fixed in 10.00% formalin. Three fragments underwent vitrification-thawing for two weeks. Two of these vitrified-thawed fragments were auto-transplanted into separate locations within the opened gluteus muscle, while the final fragment was fixed in 10.00% formalin. Eight weeks after re-implantation, biopsies were collected from the ovarian fragments and fixed in 10.00% formalin. The numbers and areas (µm²) of morphologically normal follicles were evaluated on sections stained with Hematoxylin and Eosin through light microscopy. The follicular morphology in the ovarian fragments immediately after vitrification-thawing remained similar to that of the fresh ovary. The number of morphologically intact follicles was significantly lower in the ovarian fragments eight weeks after re-implantation than fresh ovary and ovarian tissue fragments after vitrification-thawing procedure. However, follicular development to the antral stage was observed in all samples eight weeks after re-implantation. There was no statistical difference in the areas (µm²) of primordial, primary, pre-antral, and antral follicles in the ovarian fragments before cryopreservation, immediately after vitrification-thawing, and following re-implantation procedures. Auto-transplantation of ovarian tissue into the gluteus muscle of rabbits could be a viable approach for preserving ovarian follicles after vitrification-thawing procedure.

Abstract Linguatula serrata can infect most ruminants and cause accidental infections in humans. It is a causative parasite of linguatulosis, a disease that not only produces economic losses in cattle but also represents a public health risk due to its zoonotic nature. This study aimed to explore the clinical and pathological findings of pulmonary linguatulosis in a rabbit. The most striking clinical findings in the deceased rabbits were wheezing and labored breathing. Grossly, the most prominent morphological changes in the lungs were well-circumscribed, flat or slightly raised, solitary grayish-white nodular lesions, and consolidated areas. The characteristically tongue-shaped developmental forms of parasites were observed on the cut surface of the lung. Histopathologically, the most noticeable morphological changes in the lung parenchyma were diffuse thickening of the inter-alveolar septum, fibrinoid necrotic vasculitis, medial smooth muscle cells hypertrophy of the arteries, alveolar emphysema, longitudinal and transverse sections of L. serrata nymphs and extra-medullary hematopoietic foci (megakaryocytes). The morphological appearance of the nymphs showed multiple transverse grooves, saw-like cuticles, peri-buccal hooks and acidophilic glands. In conclusion, these findings reveal the etiopathological diagnosis of linguatulosis and suggest that the lungs might be a target organ in addition to the liver and lymph nodes.

Abstract Sustained release drug formulations are frequently developed to reduce dosage frequency and to improve outcomes of drug therapy. This study evaluates the pharmacokinetic (PK) parameters of a novel injectable danofloxacin (DANO) formulation in comparison with a conventional product in an animal model. A recently synthesized DANO formulation, prepared by incorporation of DANO-loaded mesoporous silica nanoparticles in liposomes and integration of liposomes in chitosan and β-glycerophosphate solution (lipogel) along with the conventional DANO product were injected subcutaneously (SC) in rabbits. Blood samples were collected at specific time points and DANO concentrations in plasma samples were measured. The PK parameters including maximum concentration (Cmax), time to reach Cmax (Tmax), area under the concentrationversustime curves (AUC), area under the first moment concentration-time curve (AUMC) and mean residence time (MRT) were studied by non-compartmental analyses. The values of MRT (156.00 ± 20.00 hr), AUC (15.30 ± 3.00 µg mL^-1 perhr) and Tmax (4.70 ± 1.60 hr) for lipogel formulation were higher than those of the conventional product (8.50 ± 3.60 hr, 3.70 ± 2.00 µg mL^-1 per hr and 0.80 ± 0.26 hr, respectively). However, Cmax values for lipogel formulation (0.41 ± 0.15 µg mL^-1) were significantly lower than those of the conventional drug product (0.68 ± 0.09 µg mL^-1). It was concluded that the novel DANO lipogel effectively slowed down the drug absorption and the incorporation of liposomes in hydrogel could be a useful approach to maintain the therapeutic drug level for a longer period; however, more studies are needed in this field.

Abstract The present study was designed to evaluate the effects of platelet growth factors and periosteal mesenchymal stem cells on bone healing process, radiographically. Forty male White New Zealand rabbits in five equal groups were used in this study. A 2 mm full thickness bone defect was made in left radial bone of each animal. In group A (control) the defect was left with no medical intervention. In group B the defect was covered by a fibrin membrane. In group C the defect was covered by a fibrin membrane plus platelet growth factors. In group D the defect was covered by a fibrin membrane plus periosteal mesenchymalstem cells, and in group E the defect was covered by a fibrin membrane enriched with platelet growth factors and periosteal mesenchymalstem cells. Radiological evaluation was done immediately after surgery (week 0) and then at the 1^st, 2^nd, 4^th, 6^th and 8^th weeks after operation. At the end of the eighth week, bone samples were taken to evaluate the histopathology. The radiological and histopathological observations showed a superior bone healing in the groups D and E, after eight weeks in comparison with the groups A, B and C. According to this study, it could be concluded that the platelet growth factors and periosteal mesenchymalstem cells could promote bone regeneration in long bone defects in a rabbit model.

Abstract Polyclonal antibodies against kappa light chain are used to diagnose diseases producing free light chain. The kappa and lambda light chains are products of immunoglobulin synthesis and released into the circulation in minor amounts such as serum, cerebrospinal fluid, urine and synovial fluid in normal condition. The purpose of this study was the production and purification of polyclonal immunoglobulin G (IgG) against human kappa light chains. In this study, early human IgG was purified by ion-exchange chromatography, reduced with Dithiothreitol and heavy and light chains were separated with size-exclusion chromatography. Afterward, affinity chromatography with protein L Sepharose at pH 2.00 was displayed to be a dominant condition for the separation and purification of the kappa light chain of immunoglobulins from human serum. Eventually, the rabbit was immunized by human kappa light chains. The rabbit IgG was purified and labeled with horseradish peroxidase (HRP). Direct enzyme-linked immunosorbent assay was planned to determine the titer of HRP conjugated rabbit IgG against the human kappa light chain. The optimum titer of anti-kappa IgG was 1:16000. At the result, purified polyclonal anti-kappa is useful tool in biomedical and biochemical researches and diagnostic kits.

Abstract Chitosan bears numerous properties, such as biocompatibility, biodegradability and non-toxicity making it suitable for use in different biomedical fields. Zinc (Zn) is required for fibroblasts proliferation and collagen synthesis as essential elements of wound healing. Its nanoparticles are well known for their capability to enhance wound healing by cell adhesion and migration improvement through growth factors-mediated mechanisms. Poor blood supply and unique histological characteristics of tendon make its regeneration always slow. Also, adhesion formation between tendon and its surrounding tissues is another problem for neotendon to return to its normal structure and functional activities. In this study, a novel tubular scaffold of zinc oxide (ZnO) nanoparticles loaded chitosan has been fabricated for tendon repair. Experimental complete tenotomy of deep digital flexor tendon in a rabbit model was done and scaffolds were placed in the transected area after two ends suturing. After four and eight weeks, adhesion formation around the tendons and tissue reaction to the scaffolds were evaluated macroscopically. Inflammation, angiogenesis and collagen fibers arrangement were also analyzed in histopathological evaluations. After eight weeks, the scaffolds were absorbed completely, adhesions around the tendon were decreased and there was no sign of significant tissue reaction and/or infection in histopathological analyses. The reduced adhesion formation, improved gliding function and better histopathological characteristics suggest this scaffold application as a potential therapy in treatment of tendon acute injuries.

Abstract Tendon never restores the complete biological and mechanical properties after healing. Several techniques are available for tissue-engineered biological augmentation for tendon healing like stem cells. Recently, synovium has been investigated as a source of cells for tissue engineering. In the present study, we investigated potentials of fibroblast like synoviocytes (FLSs) in tendon healing. Sixteen rabbits were divided randomly into control and treatment groups. One rabbit was used as a donor of synovial membrane (synovium). The injury model was unilateral complete transection through the middle one third of deep digital flexor tendon (DDFT). Subsequently, the tendon stumps were sutured with 3/0 nylon. In treatment group, 0.1 mL phosphate-buffered saline (PBS) solution containing 1 × 10⁶ nucleated cells of FLSs was injected intratendinously at both tendon stumps just next to incision line. In control group, 0.1 mL PBS without FLSs was used for intratendinous injection. Model animals were euthanized at eight weeks, DDFTs were harvested and prepared for biomechanical study. Results of study showed that, there was no significant differences in biomechanical parameters values between FLSs treated and control groups. In conclusion, intratendinous injection of FLSs did not improve biomechanical properties during eight weeks in rabbit.

Abstract This prospective study aimed to compare the intraosseous (IO) and intravenous (IV) effects of propofol on selected blood parameters and physiological variables during general anesthesia in rabbits. Thirty New Zealand White rabbits were studied. Six rabbits received IV propofol (group 1) and another 6 rabbits, were injected propofol intraosseously (Group 2) for 30 minutes (experimental groups). Rabbits of the third and fourth groups received IV and IO normal saline at the same volume given to the experimental groups, respectively. In the fifth group IO cannulation was performed but neither propofol nor normal saline were administered. Blood profiles were assayed before induction and after recovery of anesthesia. Heart and respiratory rates, rectal temperature, saturation of peripheral oxygen and mean arterial blood pressure were recorded. Heart rate increased significantly 1 to 5 minutes after induction of anesthesia in experimental groups (P < 0.05). Although mean arterial blood pressure decreased significantly from baseline, values remained above 60 mm Hg (P < 0.05). Respiratory rate decreased significantly in experimental groups, but remained higher in group 2 (P < 0.05). The lymphocyte count decreased significantly in group 1 (P < 0.05). The concentration of alkaline phosphatase in all rabbits, aspartate aminotransferase and gamma-glutamyl transferase in the first group and gamma-glutamyl transferase in the third group increased significantly (P < 0.05). Total bilirubin decreased significantly in group 2 (P < 0.05). All measured values remained within normal limits. Based on the least significant physiological, hematological and biochemical effects, the IO injection of propofol appears to be safe and suitable method of anesthesia in rabbits with limited vascular access.

Abstract Oxygen is an essential part of the most important metabolic pathways in aerobic organisms. Oxygen delivery is merely dependent on blood, rendering blood loss a devastating event. Traumatic pre-hospital liver bleeding is a major cause of early trauma deaths in human and animals, with no established therapeutic method yet. Increasing intra-abdominal pressure (IAP) has been shown to reduce liver bleeding by half. Although reduction of blood loss could be in favor of blood oxygen delivery, however, the complex interaction between increased IAP and respiratory mechanics during severe hemorrhagic shock remained unclear. We used a novel model of liver trauma in 16 rabbits and randomly assigned them to either normotensive abdomen group or increased IAP by fluid infusion (HA) groups (n=8 each). Liver size and the amount of liver injury were evaluated. Various blood oxygenation parameters were recorded. Both groups were identical in terms of the liver size and injury. The HA group had significantly lower shock index. Arterial oxygen capacity and oxygen content were higher in the HA group. No significant statistical difference was seen between groups in terms of abdominal perfusion pressure; alveolar pressure of oxygen; dissolved oxygen in blood plasma; alveolar to arterial oxygen tension gradient; arterial to alveolar oxygen pressure ratio; the ratio between partial pressure of arterial oxygen and fraction of inspired oxygen; and respiratory index. In conclusion, the novel therapeutic method of increasing IAP by fluid infusion in a rabbit model of liver hemorrhage preserved blood oxygenation better than the classic therapeutic method.

Abstract Tendon never restores the complete biological and mechanical properties after healing. Bone marrow and recently adipose tissue have been used as the sources of mesenchymal stem cells, which have been proven to enhance tendon healing. Stromal vascular fraction (SVF), derived from adipose tissue by an enzymatic digestion, represents an alternative source of multipotent cells, which undergo differentiation into multiple lineages to be used in regenerative medicine. In the present study, we investigated potentials of this source on tendon healing. Twenty rabbits were divided into control and treatment groups. Five rabbits were used as donors of adipose tissue. The injury model was unilateral complete transection through the middle one third of deep digital flexor tendon. Immediately after suture repair, either fresh stromal vascular fraction from enzymatic digestion of adipose tissue or placebo was intratendinously injected into the suture site in treatments and controls, respectively. Cast immobilization was continued for two weeks after surgery. Animals were sacrificed at the third week and tendons underwent histological, immunohistochemical, and mechanical evaluations. By histology, improved fibrillar organization and remodeling of neotendon were observed in treatment group. Immunohistochemistry revealed an insignificant increase in collagen type III and I expression in treatments over controls. Mechanical testing showed significant increase in maximum load and energy absorption in SVF treated tendons. The present study showed that intratendinous injection of uncultured adipose derived stromal vascular fraction improved structural and mechanical properties of repaired tendon and it could be an effective modality for treating tendon laceration.

Abstract Ultrasonographic examination of urinary system (kidney and urinary bladder) was conducted in New Zealand white rabbit [NZwr] and Tolai hare (Lepus tolai). Ultrasound images of the kidney and urinary bladder were evaluated on fifteen healthy rabbits of New Zealand white rabbit and another fifteen Tolai hares. The healthy rabbits were 8-12 months old (mean = 9.3 months), of both sexes and weighed between 1.1-1.7 kg (mean = 1.250 kg). All examinations were performed while the rabbits were in dorsal recumbancy. The kidneys were examined from fossa by the use of an 8 MHz linear real-time scanner. This study revealed the following measurements normal rabbit kidneys: 27.80-35.70 mm and 16.90-22.40 mm in length and width in New Zealand white rabbit, respectively. The length and width were 26.67-34.50 and 15.82-20.60 mm, in Tolai hare, respectively. Bladder wall thickness varies from 1.70-2.50 mm (in New Zealand white rabbit) to 1.80-2.60 mm (in Tolai hare). Statistical analysis showed that the gender did not have effect on length, width and weight (P > 0.05), but the type of the animal, had significant effect on the cortex and surface (P < 0.05). In the present study, the renal cortex was uniform in echogenicity, hyperechoic to the renal medulla, hypoechoic to the spleen, and isoechoic to the hepatic parenchyma.

Abstract Rabbits are widely used as laboratory animals for experimental surgery. Anesthesia of rabbits may present complications unless the method is easy to apply and safe to use. In present study, effects of different dosages of vitamin C on thiopental sodium induced anesthesia in 25 male New Zealand white rabbits were studied. In the animals that had not received vitamin C treatment before thiopental sodium induced general anesthesia, return mean time of front limb pedal, corneal and ear pinch reflexes were 6.40 ± 1.67, 6.60 ± 2.96 and 8.00 ± 2.58 minutes, respectively. Pre-treatment of rabbits with 30 and 240 mg kg-1 (IV) of vitamin C followed by thiopental sodium 20 mg kg-1 (IV) resulted in significant (P < 0.05) increase in front limb pedal reflex return mean time to 13.00 ± 2.24 and ear pinch to 11.60 ± 4.16 minutes, respectively. There was also significant (P < 0.05) decrease in the heart rate following induction of anesthesia in the animals pre-treated with 30 and 90 mg kg-1 (IV) vitamin C and no change in the animals pre-treated with 240 mg kg-1 (IV) vitamin C. Serum analysis indicated a significant (P < 0.05) increase in blood glucose. These results suggest that premedication of rabbits with vitamin C despite potentiating of thiopental sodium anesthesia in rabbits is not dose dependent.

Abstract Camel milk has an importance in the treatment of diabetes. It has been shown that the patients who drink camel milk daily, their need to insulin decrease. Therefore, this study aimed to investigate the effect of camel milk in comparison with insulin treatment in experimentally-induced diabetes. This study was carried out on forty male New Zealand rabbits, divided into four groups with ten rabbits in each. The first group G1 was considered as control non-diabetic group and received only normal saline solution. The other animals were injected intravenously with alloxan for induction of diabetes mellitus and then divided into three groups' ten rabbits each as the follows: G2 considered as control diabetic and left untreated, G3 was considered as diabetic and treated with insulin, and G4 was considered as diabetic and received camel milk. At the end of the experiment (4 weeks), blood (whole blood & serum) and tissue samples (liver, kidney and pancreas) were collected from all the animals for analysis of: enzymatic SOD and catalase, non-enzymatic GSH antioxidant enzyme activities. Serum malondialdeyde, glucose, insulin and lipid profile also were analyzed. The results showed that the camel milk was effective in the treatment of diabetes in comparison to insulin treatment alone. In addition to its hypoglycemic effect, camel milk improved the diabetes-induced oxidative stress. The histopathological evaluations demonstrated that there was a regeneration in β cells and the islets of Langerhans among the pancreatic acini in rabbits receiving camel milk. Our findings suggested that the camel milk administration in case of insulin dependant diabetes mellitus might be recommended as an oral anti-diabetic remedy.