Methods to Detect Apoptosis in Equine Peripheral Blood Neutrophils from Normal Healthy Adult Horses
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Apoptosis is a form of "planned cell death" and is an essential component of normal tissue differentiation and functional regulation. Neutrophil apoptosis facilitates down regulation of the inflammatory response while minimizing "by stander" injury to normal tissue, and disruption of this process by various diseases may have a significant negative impact on patient recovery. Consequently, neutrophil apoptosis has been the focus of research in many species. However, methods for measuring apoptosis have not been evaluated in the horse. The goal of this study was to adapt previously reported methods for inducing and measuring both neutrophil apoptosis and necrosis in non-equine species for use in equine peripheral blood neutrophils. To achieve this goal the experiment was divided into three parts: 1. Induce apoptosis and necrosis in equine peripheral blood neutrophils using previously used known inducers and examine the relationship between exposure time and percentage of affected cells; 2. Measure percentage of apoptosis and necrosis using three methods of detection: a) Annexin-V Fitc PI assay, b) Homogenous caspase 3/7 assay and c) Light microscopy and; 3. Compare the results between the three methods of apoptosis detection to determine if results are comparable The hypothesis was that previously reported methods for inducing and measuring both neutrophil apoptosis and necrosis in non-equine species can be adapted for use in equine peripheral blood neutrophils. Venous blood samples were collected aseptically from the jugular vein of eight horses. Isolation of neutrophils was performed using density gradient centrifugation on percoll. In part 1 of the experiment aliquots of the neutrophil suspension were cultured in the presence of four known inducers of apoptosis; actinomycin D, staurosporin, cycloheximide and sodium hypochlorite, at four different concentrations (table 2). A fifth population was to induce necrosis using a freeze-thaw cycle and bleach. A control sample was examined (no inducer) to determine spontaneous rate of apoptosis. The aliquots were cultured and the percentage of apoptosis determined at two sequential time points for each horse. Apoptosis was measured at either 30 minutes and 3 hours or 6 and 12 hours by three simultaneous methods: (1) annexin-V FITC PI assay (AVF), (2) homogenous caspase assay (HC) and (3) light microscopy (MS). The AVF and HC methods detect events associated with early apoptosis whilst MS detects nuclear changes which are late events of apoptosis. Using AVF and MS apoptotic cells are able to be differentiated from necrotic cells. In part two of the experiment the agreement and reproducibility between AVF and MS was further examined. In this part of the experiment neutrophils were isolated from the peripheral blood of 10 normal healthy adult horses. Each isolated sample was cultured with 80µM Actinomycin D for 12 hours and a control sample (no inducer) also prepared. Three triplicate samples were next set up from both the induced and control sample and apoptosis was determined using both AVF and MS. In part 3 of the experiment, data was analyzed using the mixed model ANOVA following log transformation of the data. Main effects of treatment, concentration and time were analyzed. Statistical significance was considered if P was < 0.05. The relationship between the three techniques; light microscopy, flow cytometry and the fluorescent plate reader, was investigated using Spearman rank correlation coefficients (Fisher's Z transformation). The Bland-Altman approach for method analysis was used to further characterize the correlation between results obtained via light microscopy and flow cytometry. Statistical significance was considered if P < 0.05. All inducers increased the percentage of apoptotic cells at either one or more time point and results were most comparable between AVF and MS. Increasing exposure time increased percentage of apoptotic neutrophils for all inducers using AVF and MS (p<0.0001). For both AVF and MS, cycloheximide and staurosporin induced apoptosis significantly above control levels at 3, 6 and 12 hours; actinomycin D at 6 and 12 hours and bleach at 3 and 6 hours as well was 12 hours for AVF only. With HC induction of apoptosis was detected earlier with bleach at 30 minutes and 3 hours and staurosporin at 30 minutes, 3 and 6 hours. Apoptosis was detected only at 6 hours for cycloheximide. Increasing concentration of inducer significantly increased the percentage apoptotic cells for staurosporin and cycloheximide between the lowest and highest concentration using AVF (p<0.001). For both AVF and MS, increasing concentration of bleach decreased the percentage of apoptotic cells (p<0.05). Increasing the concentration of staurosporin resulted in an increase in apoptosis at 30 minutes and 3 hours. Both bleach and the freeze-thaw cycle induced necrosis at all time periods excluding 30 minutes for the freeze-thaw cycle (p<0.0001). Spearman rank correlation coefficients revealed a very high correlation for percentage apoptosis and necrosis between AVF and MS (r2 = 0.91, 95% CI 0.89 – 0.93). A high correlation was also present for AVF and HC (r2 = 0.75, 95% CI 0.69 – 0.79) and MS and HC (r2 = 0.76, 95% CI 0.71 – 0.81). The lower limit of the confidence intervals suggests there is some concern about the similarity between AVF, HC and MS, HC. The Bland and Altman statistical approach indicates that both AVF and MS are highly reproducible methods with minimal variation between the triplicate samples (AVF: 8.9%, 95% CI 6.25 – 11.6%, MS 7.9%, 95% CI 6 – 9.8%). The mean difference between the two methods is 6.7% (95% CI 3.89 – 9.42%). The 95% limits of agreement indicate that results from MS can be 8.7% below to 22% above results from AVF (95% CI -13.41 – 26.7%). These findings indicate that caspase activation may occur prior to phosphatidylserine externalization and visible nuclear changes, which is in accordance with previously published data. We discovered that actinomycin D induces significant and reproducible equine peripheral blood neutrophil apoptosis in a time dependant fashion. Similarly, necrosis results from a freeze-thaw cycle or high concentration of bleach and is suitable as a positive control for necrosis. Apoptosis was effectively detected using AVF assay and results indicate good correlation between AVF and MS with an acceptably low mean difference. MS could serve as an inexpensive, simple and quick on site method to rapidly verify results attained from AVF. Induction of apoptosis using the HC was not consistent and can not be recommended based on the results of this study. Future investigation aimed at evaluating assays multiplexed to the AVF which detect other aspects of the apoptotic pathway would lead to increased confidence of results and further evidence of the mode of cell death prior to undertaking clinical studies.
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