Estimation of the postmortem interval (PMI) is a poorly studied field in veterinary pathology. The development of field-applicable methods is needed given that animal cruelty investigations are increasing continually. We evaluated various histologic criteria in equine brain, liver, and muscle tissue to aid the estimation of PMI in horses, which is central to forensic investigations of suspicious death. After death, autolysis proceeds predictably, depending on environmental conditions. Currently, no field-applied methods exist that accurately estimate the PMI using histology in animals or humans through quantification of autolysis. Brain, liver, and skeletal muscle from 12 freshly euthanized horses were held at 22°C and 8°C for 72 h. Tissues were sampled at T0h, T1h, T2h, T4h, T6h, T12h, T24h, T36h, T48h, T60h, and T72h. For each tissue, we quantified 5 to 7 criteria associated with autolysis, based on the percentage of microscopic field involved. Each criterion was modeled, with temperature and time as independent variables. Changes were most predictable in liver and muscle over the first 72 h postmortem. The criteria for autolysis that were present most extensively at both temperatures were hepatocyte individualization and the separation of bile duct epithelium from the basement membrane. The changes that were present next most extensively were disruption of myofiber continuity, hypereosinophilia, and loss of striation. Brain changes were highly variable. The high statistical correlation between the parameter “autolysis” and the variables “time/temperature”, indicates that autolysis is progressive and predictable. Further investigation of these criteria is needed to establish histologic algorithms for PMI.

Phenotypic characterization of cellular responses in equine infectious encephalitides has had limited description of both peripheral and resident cell populations in central nervous system (CNS) tissues due to limited species-specific reagents that react with formalin-fixed, paraffin embedded tissue (FFPE). This study identified a set of antibodies for investigating the immunopathology of infectious CNS diseases in horses. Multiple commercially available staining reagents and antibodies derived from antigens of various species for manual immunohistochemistry (IHC) were screened. Several techniques and reagents for heat-induced antigen retrieval, non-specific protein blocking, endogenous peroxidase blocking, and visualization-detection systems were tested during IHC protocol development. Boiling of slides in a low pH, citrate-based buffer solution in a double-boiler system was most consistent for epitope retrieval. Pressure-cooking, microwaving, high pH buffers, and proteinase K solutions often resulted in tissue disruption or no reactivity. Optimal blocking reagents and concentrations of each working antibody were determined. Ultimately, a set of monoclonal (mAb) and polyclonal antibodies (pAb) were identified for CD3+ (pAb A0452, Dako) T-lymphocytes, CD79αcy+ B-lymphocytes (mAb HM57, Dako), macrophages (mAb MAC387, Leica), NF-H+ neurons (mAb NAP4, EnCor Biotechnology), microglia/macrophage (pAb Iba-1, Wako), and GFAP+ astrocytes (mAb 5C10, EnCor Biotechnology). In paraffin embedded tissues, mAbs and pAbs derived from human and swine antigens were very successful at binding equine tissue targets. Individual, optimized protocols are provided for each positively reactive antibody for analyzing equine neuroinflammatory disease histopathology.

Phenotypic characterization of cellular responses in equine infectious encephalitides has had limited description of both peripheral and resident cell populations in central nervous system (CNS) tissues due to limited species-specific reagents that react with formalin-fixed, paraffin embedded tissue (FFPE). This study identified a set of antibodies for investigating the immunopathology of infectious CNS diseases in horses. Multiple commercially available staining reagents and antibodies derived from antigens of various species for manual immunohistochemistry (IHC) were screened. Several techniques and reagents for heat-induced antigen retrieval, non-specific protein blocking, endogenous peroxidase blocking, and visualization-detection systems were tested during IHC protocol development. Boiling of slides in a low pH, citrate-based buffer solution in a double-boiler system was most consistent for epitope retrieval. Pressure-cooking, microwaving, high pH buffers, and proteinase K solutions often resulted in tissue disruption or no reactivity. Optimal blocking reagents and concentrations of each working antibody were determined. Ultimately, a set of monoclonal (mAb) and polyclonal antibodies (pAb) were identified for CD3+ (pAb A0452, Dako) T-lymphocytes, CD79αcy+ B-lymphocytes (mAb HM57, Dako), macrophages (mAb MAC387, Leica), NF-H+ neurons (mAb NAP4, EnCor Biotechnology), microglia/macrophage (pAb Iba-1, Wako), and GFAP+ astrocytes (mAb 5C10, EnCor Biotechnology). In paraffin embedded tissues, mAbs and pAbs derived from human and swine antigens were very successful at binding equine tissue targets. Individual, optimized protocols are provided for each positively reactive antibody for analyzing equine neuroinflammatory disease histopathology.

The data show that WNV-challenged horses recruit a mixed T cell population at the onset of neurologic disease.

No abstract

2016-12-23T18:49:38

Phenotypic characterization of cellular responses in equine infectious encephalitides has had limited description of both peripheral and resident cell populations in central nervous system (CNS) tissues due to limited species-specific reagents that react with formalinfixed, paraffin embedded tissue (FFPE). This study identified a cassette of antibodies for investigating the immunopathology of infectious CNS diseases in horses. Multiple commercially available staining reagents and antibodies derived from antigens of various species for manual immunohistochemistry (IHC) were screened. Several techniques and reagents for heat-induced antigen retrieval, non-specific protein blocking, endogenous peroxidase blocking, and visualization-detection systems were tested during IHC protocol development. Boiling of slides in a low pH, citrate-based buffer solution in a double-boiler system was most consistent for epitope retrieval. Pressure-cooking, microwaving, high pH buffers, and proteinase K solutions often resulted in tissue disruption or no reactivity.Optimal blocking reagents and concentrations of each working antibody were determined.Ultimately, a cassette of monoclonal (mAb) and polyclonal antibodies (pAb) were identified for CD3 + (pAb A0452, Dako) T-lymphocytes, CD79αcy + B-lymphocytes (mAb HM57, Dako), macrophages (mAb MAC387, Leica), NF-H + neurons (mAb NAP4, EnCor Biotechnology), microglia/macrophage (pAb Iba-1, Wako), and GFAP + astrocytes (mAb 5C10, EnCor Biotechnology). In paraffin embedded tissues, mAbs and pAbs derived from human and swine antigens were very successful at binding equine tissue targets. Individual, optimized protocols are provided for each positively reactive antibody for analyzing equine neuroinflammatory disease histopathology. , 66 Bourgeois et al., 2011, Rushton et al., 2013, and Yu et al., 2015. Cell marker panels are often 67 composed of both equine and non-equine specific antibodies, of which most are used in flow 68 cytometry. Since manufacturers may not have supporting technical documentation on whether 69 their products will cross-react with equine antigens (Beckstead, 1994 andRamos-Vara, 2005), 70 development of antibody panels to accomplish in situ disease characterization in formalin-fixed 71 tissue is a formidable task because cross-linking of antigens often renders epitopes non-reactive. (Beckstead, 1994, Gutierrez et al., 88 1999, Porter et al., 2003, and Seino et al., 2007 96 Tissue processing 97 The invariable IHC procedures for all protocols included sectioning FFPE tissues at 5 m and 98 placing them on positively charged glass slides. The slides were soaked in xylene (Fischer 99 Scientific, Pittsburg, PA, USA) three times for 5 min to remove paraffin. These sections were 100 then rehydrated through a gradient of ethanol (Fischer Scientific) for 5 min in each 101 concentration, 100%, 100%, 95%, and 70% ethanol, followed by de-ionized water. In order to 102 reduce the volume of the reagents tested and liquid loss, tissues were encircled with a 103 hydrophobic barrier...