nan Amarpal

Isolation, culture and characterization of caprine mesenchymal stem cells derived from amniotic fluid

Amniotic fluid (AF) represents heterologous cell types and a specific group of these cells show high growth rate and multipotent characteristics. The aim of the present study was to culture and fully characterize the putative stem cell population isolated from caprine mesenchymal stem cells. Plastic adherent fibroblastoid cell population could be successfully isolated from the caprine amniotic fluid. In vitro expanded caprine amniotic fluid derived mesenchymal stem cells (cAF-MSCs) showed high proliferation ratio with a doubling time of 33.1h and stained positive for alkaline phosphatase. Relative transcript abundance of CD-73, CD-90 and CD-105 surface markers were analyzed by SYBR green based real time PCR and their respective proteins were localized through immunocytochemistry, however cAF-MSCs were found negative for haematopoietic marker CD-34. When exposed to corresponding induction condition, cAF-MSCs differentiated into osteogenic, adipogenic and chondrogenic lineages which was confirmed through von Kossa, Oil Red O and Alcian blue staining respectively. Furthermore, these cells were found positive for undifferentiated embryonic stem cell markers like Oct-4, Nanog, Sox-2, SSEA-1 and SSEA-4 which accentuate their pluripotent property. In conclusion, caprine amniotic fluid represents a promising source of mesenchymal stem cells with high proliferative and differentiation potential and these cells offer their scope for multiple regenerative therapies.

Comparison of autogenic and allogenic bone marrow derived mesenchymal stem cells for repair of segmental bone defects in rabbits.

Autogenic and allogenic bone marrow derived mesenchymal stem cells (BM-MSCs) were compared for repair of bone gap defect in rabbits. BM-MSCs were isolated from bone marrow aspirates and cultured in vitro for allogenic and autogenic transplantation. A 5mm segmental defect was created in mid-diaphysis of the radius bone. The defect was filled with hydroxyapatite alone, hydroxyapatite with autogeneic BM-MSCs and hydroxyapatite with allogenic BM-MSCs in groups A, B and C, respectively. On an average 3.45×10(6) cells were implanted at each defect site. Complete bridging of bone gap with newly formed bone was faster in both treatment groups as compared to control group. Histologically, increased osteogenesis, early and better reorganization of cancellous bone and more bone marrow formation were discernible in treatment groups as compared to control group. It was concluded that in vitro culture expanded allogenic and autogenic BM-MSCs induce similar, but faster and better healing as compared to control.

Evaluation of autologous bone marrow‐derived nucleated cells for healing of full‐thickness skin wounds in rabbits

The aim of the study was to evaluate the potential of autologous bone marrow‐derived nucleated cells to enhance the rate of healing of full‐thickness excisional skin wounds in rabbits. The study was conducted on 20 New Zealand white rabbits of either sex. Two, 2 × 2 cm full‐thickness skin (thoracolumabar region) excisional wounds were created; one on each side of the dorsal midline in each animal. The wounds were randomly assigned to either injection of autologous bone marrow‐derived nucleated cells into the wound margins (BI), or topical application of sterile saline solution (normal saline, NS), which served as control. The wound healing was assessed by evaluation of granulation tissue formation, wound contraction, epithelisation and histopathological and histochemical changes up to 28 days after creation of the wound. Granulation tissue appeared significantly faster in BI‐treated wounds (3.22 ± 0.22 days) than in NS‐treated wounds (4.56 ± 0.47 days). Better epithelisation was seen histologically in BI wounds than in NS‐treated wounds. Wound contraction was significantly more in BI wounds when compared with NS wounds on 21 post‐surgery. Histopathological examination of the healing tissue showed early disappearance of inflammatory reaction, significantly more neovascularisation, and more fibroplasias and early lay down and histological maturation of collagen in BI wounds than in control wounds. It was concluded that injection of autologous bone marrow‐derived nucleated cells in the wound margins induced faster and better quality healing of excisional skin wounds in rabbits when compared with normal saline. The injection of autologous bone marrow‐derived nucleated cells can be used to promote healing of large full‐thickness skin wounds in rabbits.

Medetomidine with ketamine and bupivacaine for epidural analgesia in buffaloes.

The efficacy of ketamine and bupivacaine in enhancing the epidural analgesia induced by medetomidine was evaluated in 10 buffalo calves utilized repeatedly after a gap of 10 days so that each drug combination was tested in 4 randomly selected animals. In group A, medetomidine (15 μg/kg), in group B ketamine (2.0 mg/kg), in group C bupivacaine (0.125 mg/kg), in group D medetomidine and ketamine (15 μg/kg and 2.0 mg/kg), and in group E medetomidine and bupivacaine (15 μg/kg and 0.125 mg/kg) was administered epidurally.Onset of analgesia was significantly earlier in animals of groups B and D compared to the animals of groups A, C and E. Medetomidine alone or in combination with ketamine/bupivacaine produced complete analgesia of the tail, perineum, inguinal region and upper parts of hind limbs. Ketamine produced a very short duration of complete analgesia at the tail and perineum. Bupivacaine alone produced only mild to moderate analgesia. Both ketamine and bupivacaine prolonged the duration of analgesia. Motor incoordination was mild to moderate in animals of all the groups, but animals remained standing throughout the period of observation. Animals of groups A, D and E showed mild to moderate sedation during the observation period. Ruminal movements decreased nonsignificantly in animals of groups A and E. Mild salivation was observed in animals of all the groups except group C.Significant decrease in heart rate (HR) was recorded after epidural administration of medetomidine or bupivacaine; however, ketamine caused short duration of tachycardia. The administration of ketamine with medetomidine caused lesser decrease in HR compared to medetomidine alone or in combination with bupivacaine. Significant fall in respiratory rate (RR) was recorded after epidural administration of medetomidine or bupivacaine alone, but an increase in RR was recorded after ketamine administration. The fall in RR was less pronounced in animals in which medetomidine was used with ketamine compared to the animals in which medetomidine was used alone or in combination with bupivacaine.Mean arterial pressure (MAP) decreased and central venous pressure (CVP) increased significantly after epidural administration of medetomidine in combination with ketamine or bupivacaine. The ECG changes included tall T wave, QS pattern, RS pattern and ST elevation and heart blocks at different intervals, which were more frequent and pronounced in animals given bupivacaine with medetomidine.It can be concluded that epidural administration of medetomidine can produce complete analgesia of the tail, perineum, inguinal region and upper hind limbs in buffaloes. However, significant depression of cardiovascular parameters was recorded. Administration of ketamine along with medetomidine resulted in significantly early onset and slightly longer duration of analgesia with lesser cardiopulmonary side-effects compared to medetomidine alone or medetomidine with bupivacaine. Addition of ketamine to medetomidine thus seems to be useful for producing epidural analgesia; however, addition of bupivacaine failed to provide any advantage over medetomidine alone.

Comparison of two doses of ropivacaine for lumbosacral epidural analgesia in buffalo calves (Bubalus bubalis)

Six male buffalo calves aged from six to eight months and weighing 100 to 120 kg were used to evaluate the efficacy and safety of caudal epidural analgesia produced by doses of 37·5 mg (5 ml) and 75 mg (10 ml) ropivacaine (0·75 per cent). The study was completed in two phases. In phase 1, all the animals were used twice to evaluate the analgesic and clinical effects of two doses of ropivacaine. In phase 2, the animals were divided into two groups of three to evaluate the effects of two doses of ropivacaine on some haemodynamic and acid-base parameters. Signs of analgesia, as evidenced by a loss of response to pinprick stimulation, were recorded only at the tail, perineum and hindlimbs of the animals given 5 ml ropivacaine, but they extended from the tail to the thorax (T9) in the animals given 10 ml ropivacaine. The duration of analgesia was five to six hours in the animals given 5 ml ropivacaine and seven to eight hours in the animals given 10 ml ropivacaine. Animals of both groups became recumbent, but the score for ataxia was higher in the animals given 10 ml ropivacaine. The respiratory rate decreased significantly (P<0·05) below the baseline in the animals given 10 ml ropivacaine, but the rectal temperature, heart rate, mean arterial pressure, central venous pressure and acid-base and electrolyte parameters did not change significantly in either group.

Management of fractures near the carpal joint of two calves by transarticular fixation with a circular external fixator.

A four-ring circular external skeletal fixation device was evaluated for transcarpal fixation of compound fractures in two calves. Case 1 was an eight-month-old female Holstein-Friesian × indigenous breed calf weighing 72 kg, which had a Salter-Harris type II fracture at the distal metaphysis of the right radius/ulna with an open contaminated wound on the medial aspect of the carpus. Case 2 was an 18-month-old crossbred Haryana heifer weighing 105 kg, which had a comminuted fracture at the proximal end of its left metacarpus with severe soft tissue trauma and an open wound on the medial aspect. In both cases, the fractures were repaired with four-ring circular fixators by fixing the proximal two rings in the distal radius/ulna and the distal two rings in the metacarpus. Postoperatively, both calves were treated with analgesic and anti-inflammatory drugs and antibiotic, and the pin-bone interfaces and the open wound were cleaned regularly with povidone-iodine solution. In both animals weight bearing was good to excellent in the immediate postoperative period. The fixator was well maintained and tolerated by the animals until the fractures healed after 45 to 60 days. The movement of the carpal joint was slightly affected when the fixator was removed on day 60, but a follow-up examination after one year showed that both calves had normal functional usage of the limbs.

Effect of bupivacaine on epidural analgesia produced by xylazine or medetomidine in buffaloes (Bubalus bubalis)

OBJECTIVEnTo evaluate and compare the effect of epidural bupivacaine on analgesia produced by epidural xylazine or medetomidine in buffaloes.nnnSTUDY DESIGNnProspective, blinded study.nnnANIMALSnTen male buffalo calves (6-8 months of age; body weight 70-90 kg) were used on two occasions to conduct a total of 20 investigations.nnnMETHODSnCaudal extradural analgesia was produced in four buffalo calves each by the injection of either xylazine (0.05 mg kg(-1)), medetomidine (15 microg kg(-1)) or 0.5% bupivacaine (0.125 mg kg(-1)), or combinations of xylazine and bupivacaine (0.05 and 0.125 mg kg(-1)), or medetomidine and bupivacaine (15 microg kg(-1) and 0.125 mg kg(-1)) at the first intercoccygeal extradural space. Analgesia was tested using deep pinprick stimuli.nnnRESULTSnExtradural administration of xylazine or medetomidine resulted in complete analgesia of the tail, perineum, inguinal region and the upper parts of the hind limbs, which was faster in onset and longer in duration in the medetomidine group than in the xylazine group. Addition of bupivacaine increased the intensity of the analgesia produced by xylazine, but not that produced by medetomidine. All the drugs caused mild to moderate ataxia, but signs of sedation were apparent only in animals which received xylazine or medetomidine. The extradural injections of all the drugs caused significant decrease in heart rate (p = 0.024), respiratory rate (p = 0.026) and rectal temperature (p = 0.036) from the respective baseline values, but the differences between the groups were not significant.nnnCONCLUSIONSnMedetomidine produced a longer duration of analgesia than that produced by xylazine. Bupivacaine prolonged the analgesia produced by xylazine, but the analgesia produced by the combination of medetomidine and bupivacaine was not superior to that produced by medetomidine alone.nnnCLINICAL RELEVANCEnBupivacaine may be used to prolong the extradural analgesia produced by xylazine, but not that produced by medetomidine in buffaloes.