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  3. A novel histochemical method for the visualization of thrombin activity in the nervous system

A novel histochemical method for the visualization of thrombin activity in the nervous system

  • Neuroscience. 2016 Apr 21;320:93-104. doi: 10.1016/j.neuroscience.2016.01.065.
D Bushi 1,  O Gera 2,  G Kostenich 3,  E Shavit-Stein 4,  R Weiss 5,  J Chapman 6,  D Tanne 7
Affiliations

Affiliations

  • 1 Comprehensive Stroke Center, Department of Neurology and The J. Sagol Neuroscience Center, Chaim Sheba Medical Center, Tel HaShomer, Israel; Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel. Electronic address: doron.bushi@gmail.com.
  • 2 Comprehensive Stroke Center, Department of Neurology and The J. Sagol Neuroscience Center, Chaim Sheba Medical Center, Tel HaShomer, Israel; Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; Department of Physical Therapy, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
  • 3 Advanced Technology Center, Chaim Sheba Medical Center, Tel HaShomer, Israel.
  • 4 Comprehensive Stroke Center, Department of Neurology and The J. Sagol Neuroscience Center, Chaim Sheba Medical Center, Tel HaShomer, Israel.
  • 5 Comprehensive Stroke Center, Department of Neurology and The J. Sagol Neuroscience Center, Chaim Sheba Medical Center, Tel HaShomer, Israel; Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel.
  • 6 Comprehensive Stroke Center, Department of Neurology and The J. Sagol Neuroscience Center, Chaim Sheba Medical Center, Tel HaShomer, Israel; Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; Department of Neurology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; Robert and Martha Harden Chair in Mental and Neurological Diseases, Sackler Faculty of Medicine, Tel Aviv University, Israel.
  • 7 Comprehensive Stroke Center, Department of Neurology and The J. Sagol Neuroscience Center, Chaim Sheba Medical Center, Tel HaShomer, Israel; Department of Neurology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel.
PMID: 26851772 DOI: 10.1016/j.neuroscience.2016.01.065
Abstract

Although Thrombin has an important role in both central and peripheral nerve diseases, characterization of the anatomical distribution of its proteolytic activity has been limited by available methods. This study presents the development, challenges, validation and implementation of a novel histochemical method for visualization of Thrombin activity in the nervous system. The method is based on the cleavage of the substrate, Boc-Asp(OBzl)-Pro-Arg-4MβNA by Thrombin to liberate free 4-methoxy-2-naphthylamine (4MβNA). In the presence of 5-nitrosalicylaldehyde, free 4MβNA is captured, yielding an insoluble yellow fluorescent precipitate which marks the site of Thrombin activity. The sensitivity of the method was determined in vitro using known concentrations of Thrombin while the specificity was verified using a highly specific Thrombin Inhibitor. Using this method we determined the spatial distribution of Thrombin activity in mouse brain following transient middle cerebral artery occlusion (tMCAo) and in mouse sciatic nerve following crush injury. Fluorescence microscopy revealed well-defined Thrombin activity localized to the right ischemic hemisphere in cortical areas and in the striatum compared to negligible Thrombin activity contralaterally. The histochemical localization of Thrombin activity following tMCAo was in good correlation with the infarct areas per triphenyltetrazolium chloride staining and to Thrombin activity measured biochemically in tissue punches (85 ± 35 and 20 ± 3 mU/ml, in the cortical and striatum areas respectively, compared to 7 ± 2 and 13 ± 2 mU/ml, in the corresponding contralateral areas; mean ± SEM; p<0.05). In addition, 24 h following crush injury, focal areas of highly elevated Thrombin activity were detected in teased sciatic fibers. This observation was supported by the biochemical assay and western blot technique. The histochemical method developed in this study can serve as an important tool for studying the role of Thrombin in physiological and pathological conditions.

Keywords

crushed sciatic nerve; enzyme histochemistry; ischemic stroke; thrombin; transient middle cerebral artery occlusion.

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