1. Histologia E Embriologia Humano Eynard Pdf Gratis

BackgroundSynovitis is an inflammation-related disease linked to rheumatoid arthritis, osteoarthritis, infections and trauma. This inflammation is accompanied by immune cells infiltration which initiates an inflammatory response causing pain, discomfort and affecting the normal joint function. The treatment of synovitis is based on the administration of anti-inflammatory drugs or biological agents such as platelet rich plasma and mesenchymal stem cells. However, the evaluation and validation of more effective therapies of synovitis requires the establishment of clinically relevant animal models. BackgroundThe synovial fluid (SF) is a transudate of plasma that provides low-friction for a normal joint function. The homeostasis of SF depends on the continuous renewal from the lymphatic capillaries to the articular cavity and the transynovial filtration towards the lymphatic capillaries.

This renewal is also facilitated by physical exercise and joint flexion. The synovitis is an inflammation-related disease usually linked to rheumatoid arthritis , osteoarthritis and viral infections. Animals and experimental designEight Large White pigs were housed in the animal facility at the Minimally Invasive Surgery Centre and used for all experimental procedures. Animals aged 3 months and weighed 25–35 kg at the beginning of the study were used. All experimental protocols were approved by the Committee on the Ethics of Animal Experiments of Minimally Invasive Surgery Centre and fully complied with recommendations outlined by the local government (Junta de Extremadura) and by the Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes.All the animals were pre-immunized by subcutaneous injections of bovine serum albumin (BSA).

Local immunizations for synovitis induction were performed by intra-articular injection of BSA on the right carpal joint of each animal. The left carpal joints received an intra-articular injection of phosphate buffer saline (PBS) to be used as negative control.As an additional control group, three Large White pigs without BSA pre-immunization were included in this study. Intra-articular injections of PBS and BSA were performed in the left and the right carpal joints respectively. Anesthetics proceduresEvery procedure was done under anesthesia. For blood sampling and subcutaneous BSA injections, anesthesia was induced by intramuscular injection of 10 mg/kg ketamine hydrochloride and 0.02 mg/kg dexmedetomidine hydrochloride.

Histologia E Embriologia Humano Eynard Pdf Gratis

The animals were recovered with 0.02 mg/kg atipamezole hydrochloride. For SF sampling and arthroscopies, anesthesia was induced by the same procedure together with 2 mg/kg propofol on intravenous bolus injection, and the analgesia was performed with 3 mg/kg of tramadol. According to ethical and animal welfare concerns, all the animals received analgesic treatment with buprenorphine hydrochloride. The buprenorphine at 0.3 mg/ml was regularly administered at 0.03 ml/kg for 7 days after intra-articular injection. Immunization protocolFor animal immunizations, a solution with 20 mg/ml of BSA (Sigma-Aldrich, St. Louis, MO, USA) was prepared and passed through a 0.2 μm sterilized microfilter. An equal volume of Freund Complete Adjuvant (FCA) (Sigma-Aldrich, St.

Louis, MO, USA) was mixed with the BSA solution and emulsified. The immunization was performed by subcutaneous injection of this emulsion. A total of 0.4 ml/kg was injected on days 0, 14 and 21 (see Fig.

On day 28, a total of 0.5 ml of SF was aspirated from both joints (SF basal sample) and intra-articular immunizations of BSA were performed in the forelimbs. A total of 0.5 ml of BSA at 20 mg/ml was injected on the right carpal joint to induce the synovitis and 0.5 ml PBS on the left carpal joint (used as negative control). Isolation and phenotypic characterization of synovial fluid and peripheral blood lymphocytesSynovial fluid leukocytes (SFLs) were obtained from carpal joints. A total of 0.5–1 ml of SF was aspirated and weekly sampled for 5 weeks (see Fig. Leukocytes were counted in an automatic hematology analyzer (Mindray BC-5300 Vet, Hamburg, Germany) and SFLs were isolated by centrifugation at 900×g and used for flow cytometric analysis.

Supernatants were also collected and stored at −20 °C for cytokines determination.Peripheral blood lymphocytes (PBLs) were obtained from jugular vein blood samples. Blood sampling was performed weekly from the beginning of the study. PBLs were isolated by centrifugation over Histopaque-1077 (Sigma-Aldrich) and washed twice with PBS for cytometric analysis.For flow cytometric analysis, PBLs and SFLs were suspended in PBS containing 2% FBS. The cells were then stained with PerCP-conjugated monoclonal antibody against porcine CD4 (Mouse Anti-Pig CD4a, clone: 74–12-4, BD Pharmingen, San Jose, CA, USA) and APC-conjugated monoclonal antibody against porcine CD8 (Mouse Anti-Pig CD8a, clone: 76–2-11, BD Pharmingen). The cytometric analysis was performed as follows: 2 × 10 5 cells were incubated for 30 min at 4 °C with appropriate concentrations of monoclonal antibodies. The cells were washed and resuspended in PBS. The flow cytometric analysis was performed in a FACScalibur cytometer (BD Biosciences) after acquisition of 10 5 events.

Cells were primarily selected using forward and side scatter characteristics and fluorescence was analyzed using CellQuest software (BD Biosciences, San Jose, CA, USA). Isotype-matched negative control antibodies were used in all the experiments. Quantification of anti-BSA antibodies by ELISAIn order to quantify the anti-BSA IgG titers on immunized animals, an ELISA test was performed on plasma samples. Microplate coating was performed by an overnight incubation with BSA at 20 μg/ml. The next day, coating solution was removed and wells were washed twice with 200 μl of PBS/Tween-20 (0.05%, 7.4 pH). In order to prevent the nonspecific binding of the antibodies, the remaining protein-binding sites were blocked by adding 200 μl of BSA and the plate was incubated for 2 h at 4 °C. The plate was washed four times with 200 μl PBS/Tween-20.

Plasma samples were diluted on PBS at 1/200 and 100 μl of this dilution was added to each well. The plate was incubated for 2 h at 4 °C.

After washing four times with PBS/Tween-20, 100 μl of 1/5000 diluted horseradish peroxidase (HRP) -conjugated secondary antibody (Rabbit Anti-Pig IgG, Thermo Fisher Scientific, Waltham, MA, USA) were added to each well and the plate was incubated for 2 h at 4 °C. Again, the plate was washed four times and 100 μl of the enzyme substrate (3,3′, 5,5′-Tetramethylbenzidineor TMB, Sigma-Aldrich) were added to each well.

Two minutes later, 100 μl of 1 N HCl were added per well in order to stop the reaction. Plate absorbance was measured at 450 nm on a Synergy Mx spectrophotometer (BioTech Industries, Newton, NC, USA). Cytokine detection and measurement with multiplex technologyThe supernatants of SF were diluted 1:4 in PBS and stored at −20 °C. These supernatants were thawed and IFNα, IFNγ, IL-1b, IL-4, IL-6, IL-8, IL-10, IL-12p40 and TNFα were analyzed using a multiplexed immunoassay.

The measurements were performed according to the manufacturer’s instructions by Luminex xMAP technology using the ProcartaPlex Porcine Cytokine & Chemokine Panel 1 (eBioscience, San Diego, CA, USA; catalog number EPX090–60829-901). The concentrations of the different cytokines were expressed as pg/ml, and calculated according to a standard curve. ArthroscopiesIn order to evaluate the potential changes on the joint status, arthroscopies were performed in the carpal joints of pre-immunized animals at two weeks after intra-articular PBS or BSA injection. The arthroscope used on surgical procedures was a HOPKINS® wide angle forward-oblique telescope 30°, 2.4 mm diameter, 10 cm length (Karl Storz, Tuttlingen, Germany).A needle was used as a guide for the correct placement of the arthroscope through a small incision. A saline flux through the joint was maintained during all the procedure to provide a better visualization of the tissues.

A careful and detailed evaluation of the joint was performed and photo recorded. Finally, the 3 mm incision was closed with a 2–0 absorbable suture and every animal received antibiotic treatment (clavulanic acid + amoxicillin) during 7 days after arthroscopy. Pressure platform gait analysis functional evaluation by biomechanical analysisA 174.5 cm × 36.9 cm pressure platform (PP) (Walkway™; Tekscan, South Boston, MA, USA), composed by individual sensors with a density of 1.4 sensor/cm 2 and 9152 sensors in total, was used for the biomechanical evaluation. The sensors of the PP walkway were calibrated according to the manufacturer’s specifications.

Seven days after intra-articular injection, animals were guided to walk along the PP and after at least 5 complete passes per animal, data were analyzed. Impulse (kg x sec) and vertical maximum force (kg) were determined. Measurements were normalized to animal weights. The BSA-immunization protocol elicits an antibody and T cell response on porcine modelThe animals were subcutaneously immunized with an emulsion of BSA and FCA on days 0, 14 and 21.

During the immunization protocol, peripheral blood was weekly collected from vein and analyzed by flow cytometry to evaluate the percentage of CD4 + T cells, CD8+ T cells and their ratio. It is important to note that anti-CD4 and anti-CD8 antibodies were simultaneously used for the quantification of CD4+/CD8- and CD4−/CD8+ subsets.

The presence of anti-BSA antibodies in plasma samples was also quantified by ELISA test.The analysis of peripheral blood lymphocytes from BSA-immunized animals demonstrated that the CD8 + T cell subset showed a trend to increase (non-statistically significant) whereas both CD4 + T cell subset and CD4/CD8 ratio showed a trend to decrease (Fig. Lymphocyte subsets distribution in peripheral blood cells from BSA-immunized animals. Peripheral blood lymphocytes were weekly collected for flow cytometry analysis. Black arrows indicate the subcutaneous BSA injections and the grey arrow indicates the intra-articular injection. The graphic shows the percentage of CD4+ T cells, CD8+ T cells and their ratio. The lower boundary of the box indicates the 25th percentile and the upper boundary the 75 thpercentile. Bars above and below the box indicate the 90th and 10 th percentiles.

The line within the box marks the median ( n = 4). No statistically significant differences were found between groupsRegarding the evaluation of antibodies in plasma samples, our results demonstrated that anti-BSA IgG antibody titers were detected in all of the four animals. The antibody concentrations significantly increased when compared 7 and 14 days and remained stable from days 14 to 35 showing a maximum level at 4 weeks (Fig. Humoral response to bovine serum albumin in immunized animals. Plasma samples were weekly collected and anti-BSA IgG levels were quantified by ELISA immunoassay. In the graphic, black arrows indicate the subcutaneous BSA injections and the grey arrow indicates the intra-articular injection. Values show the mean ± SD ( n = 4).Statistically significant difference ( p ≤ 0.05) compared to basal levelBased on these immunoassays, here we demonstrate that BSA-immunization protocol triggered the pre-sensitization of this animal model, which is prerequisite to generate an antigen-induced synovitis.

Intra-articular administration of BSA on pre-immunized animals modifies the leukocyte counts and synovial lymphocytes distributionThe pre-sensitized animals (subcutaneously immunized with BSA at day 0, 14 and 21) received an intra-articular injection of PBS or BSA in left or right carpal joints, respectively, at day 28. Basal samples were aspirated at day 28 prior to PBS or BSA injections. Synovial fluids were aspirated at days 35, 42, 49 and 56 (Fig. The synovial fluids were centrifuged and synovial leukocytes were processed for flow cytometry analysis. Non-cellular fraction of synovial fluid was frozen for subsequent cytokine analyses.The counting of leukocytes from synovial fluid samples demonstrated that, at day 35 (7 days post intra-articular BSA), the leukocyte counts were significantly increased ( p = 0.04) in those carpal joints where BSA was intra-articularly injected: 0.75 ± 1.12 × 10 6 /ml in control samples vs 2.40 ± 1.19 × 10 6/ml in BSA-injected.Moreover, the lymphoid and myeloid synovial cells were quantified in an automatic hematology analyzer. The distribution of lymphoid/myeloid cells in control samples was: 67.95 ± 6.57 (% of lymphoid cells) and 32.05 ± 8.11 (% of myeloid cells). On the other hand, the distribution of lymphoid/myeloid cells after intra-articular BSA injections was: 40.9 ± 19.93 (% of lymphoid cells) and 59.1 ± 20.79 (% of myeloid cells).Once demonstrated that leukocyte counts were significantly increased, the analysis of synovial lymphocytes CD4 + T cells, CD8 + T cells and their ratio was performed at day 7, 14 and 21 after intra-articular BSA or PBS injections.

Our results did not show any significant difference at days 14 and 21 (data not shown). In contrast, significant differences were observed when synovial lymphocytes were quantified at day 7 after intra-articular BSA injections (Fig.

As shown in Fig., the intra-articular administration of BSA on pre-immunized animals exerted a significant decrease of synovial CD8 + T cells when compared to basal values ( p = 0.025). In contrast, the percentage of CD4 + T cells as well as the CD4/CD8 ratio was significantly increased ( p = 0.025 and p = 0.026, respectively).

It is important to note that, in order to have a control for intra-articular injections, PBS was intra-articularly injected in pre-sensitized animals and no significant differences were observed when compared to basal values (Fig. Moreover, in order to establish a proper negative control, PBS and BSA were intra-articularly injected in non-immunized animals. No differences were observed in terms of CD4 + T cells, CD8 + T cells, CD4/CD8 ratio (Additional file ) and biochemical parameters (Additional file ). Distribution of synovial lymphocyte subsets. Synovial fluid lymphocytes were collected for flow cytometric analysis just before intra-articular injection (basal) and 7 days after.

The graphic shows the percentage of CD4+ T cells ( a) CD8+ T cells ( b) and their ratio c. Values show the mean ± SD ( n = 3).Statistically significant difference ( p ≤ 0.05) compared to basal levelAltogether, our results demonstrated that intra-articular administration of BSA on pre-immunized animals elicited a significant increase of synovial leukocytes as well as a redistribution of synovial T cell subsets towards a CD4-driven response. Local administration of BSA on pre-sensitized animals modifies the cytokine profile of synovial fluidOnce evaluated the changes in the leukocyte counts as well as in the percentages of synovial CD4+ and CD8+ T cells, we aimed to evaluate the inflammatory environment by quantifying a wide range of cytokines.

The following cytokines were quantified by Luminex technology: IFNα, IFNγ, IL-1b, IL-4, IL-6, IL-8, IL-10, IL-12p40 and TNFα.The synovial fluids from immunized animals only showed detectable and significant differences on IL-12p40 cytokine (data not shown for the rest of cytokines). This cytokine was quantified at days 7, 14 and 21 after intra-articular BSA injections.Our results showed significant differences on IL-12p40 at day 7 after intra-articular BSA-immunization ( p ≤ 0.05) and non-significant differences (but a trend to increase) were found at day 14. Finally, non-significant differences were observed after 21 days (Fig.

Quantification of IL-12p40 levels in synovial fluid. Synovial fluid was collected at day 7, 14 and 21 after intra-articular injection of PBS and BSA. Cytokine levels were determined by Luminex xMAP technology. The lower boundary of the box indicates the 25th percentile and the upper boundary the 75th percentile. Bars above and below the box indicate the 90 th and 10 th percentiles. The line within the box marks the median ( n = 4).

Dot line indicates the basal levels (just before intra-articular injection).Statistically significant difference ( p ≤ 0.05) compared to basal level. Arthroscopy as a diagnostic procedure in synovitisCarpal joints from BSA-immunized animals were evaluated by minimally invasive procedures. Arthroscopy was performed at day 14 after intra-articular BSA or PBS injections. A total of 8 arthroscopic evaluations were performed and four out of four carpal joints where BSA was intra-articularly injected showed a slightly red to orange color (Fig.

In contrast, those control carpal joints where PBS was injected, showed clear, colorless or straw colored synovia (Fig. Download viu for pc windows 10. Surgical approach and arthroscopic analysis. Two weeks after intra-articular injection of PBS or BSA, an arthroscopic evaluation was performed. Figure shows the access to the articular cavity a, the arthroscopic procedure b representative image of arthroscopy in the control joint c and representative image of arthroscopy in the BSA-injected joint d. Synovial fluid classification according to nucleated cells/mm 3 e. Synovial fluid is classified as “normal” if it contains less than 180 nucleated cells/mm 3 or “non-inflammatory” when synovial fluid contains less than 2000 cells/mm 3. On the contrary, when synovial fluid contains 2000–50,000 cells/mm 3 it is classified as “inflammatory” Apart from the macroscopic observation, aspirated synovial fluids from control and BSA were classified according to synovial nucleated cells.

This classification is based on a previous report from El-Gabalawy where synovial fluid can be classified as Normal, if it contains fewer than 180 nucleated cells/mm 3; Non-inflammatory, when synovial fluid contains less than 2000 cells/mm 3, and Inflammatory, when synovial fluid contains 2000–50,000 cells/mm 3. Our results demonstrated that, synovial fluid from control joints can be classified as Normal or Non-inflammatory and those synovial fluids where BSA was injected could be considered as Inflammatory (Fig. Monitoring of synovitis by pressure platform gait analysisThe kinematic gait parameters were evaluated by a pressure platform. The pre-sensitized animals were biomechanically evaluated at day 7 after intra-articular injections of BSA or PBS. The parameters evaluated were impulse (Kg x sec) and the vertical maximum force (Kg).

In terms of animal management, our experience demonstrated that kinematic parameters could be easily quantified with the porcine model (Fig. Our results showed that, the impulses in the forelimbs with BSA showed an enormous inter-individual variability (Fig. ) and no significant difference was observed in terms of vertical maximum force (Fig. Finally, it is important to note that because of ethics and animal welfare, this kinematic analysis had to be performed under analgesia. Pressure platform gait analysis.

Seven days after intra-articular injection of PBS or BSA, a pressure platform gait analysis was performed to evaluate plantar pressure distributions. A Above, a representative image of the gait analysis ( LF: left forelimb; LH: left hind limb; RF: right forelimb; RH: right hind limb) is represented. Below, the pressure of each limb is shown. The legend on the right shows the equivalence between numeric and colorimetric values. Maximum forces b and impulses c in control and BSA-injected limbs ( n = 4). The lower boundary of the box indicates the 25 th percentile and the upper boundary the 75th percentile. Bars above and below the box indicate the 90 th and 10 th percentiles.

The line within the box marks the median. No significant differences were found between PBS and BSA injected joints. ConclusionsTaking into consideration that the establishment of an animal model is an absolute prerequisite in preclinical research, this paper describes the development and characterization of a clinically relevant porcine model of synovitis.

This antigen-induced inflammation model triggered a cell-mediated response allowing us the identification of immunological parameters to be used as biomarkers in the monitoring of synovitis and newly developed therapies. Moreover, here we demonstrated that, our antigen-induced model of synovitis can also be evaluated by standard arthroscopic instruments and kinetic studies.