A 6-year-old, neutered male Saint Bernard dog was presented with a 1-month history of ataxia, hyp... more A 6-year-old, neutered male Saint Bernard dog was presented with a 1-month history of ataxia, hypermetria and head tilt. High-field magnetic resonance imaging revealed a mass in the cerebellar vermis. During necropsy examination, a cream-coloured irregular area was observed in the cerebellar white matter. Microscopically, the mass comprised a diffuse neoplastic proliferation of spindle cells with oval pleomorphic nuclei in the white and grey matter of the cerebellum and pons and in the subpial area. Neoplastic infiltration was not found in the cerebrum. Immunohistochemistry revealed that the neoplastic cells were positive for vimentin and partially positive for glial fibrillary acidic protein. Based on these findings, the neoplastic lesion was diagnosed as gliomatosis cerebelli, without involvement of the cerebrum.
To study differences in the distribution of proteoglycans and their relationships to collagen fib... more To study differences in the distribution of proteoglycans and their relationships to collagen fibrils in the cornea and sclera, bovine cornea and sclera were examined histochemically using the ruthenium red (RR) staining method. RR-positive granules (30 nm in diameter) were present in association with fine filamentous materials (3 nm in diameter and 30-100 nm in length) in the interfascicular spaces of collagen bundles in both the corneal stroma and sclera. The amount of these materials was smaller in the sclera than in the cornea. The characteristic band-like arrangement of RR-positive granules connected by filamentous materials at intervals of 80-100 nm was found only in the cornea. In enzyme digestion experiments, tissue sections were treated by chondroitinase ABC, AC, and keratanase before RR staining. The RR-positive granules and their associated filamentous materials were darkly stained after chondroitinase AC or keratanase digestion, but displayed markedly lighter staining after chondroitinase ABC digestion. These results indicate that RR-positive granules and filamentous materials contain dermatan sulfate proteoglycan.
Abstract 2300 Introduction: The derivation of blood platelets from bone marrow (BM) MKs has been ... more Abstract 2300 Introduction: The derivation of blood platelets from bone marrow (BM) MKs has been known since 1906, and it evoked long dispute for the process of platelet production between proplatelet model and platelet territory model. The proplatelet model, in which MKs extend long thin protrusion into the sinusoidal lumen and the tip of proplatelet then bud off, is supported by the images from in vitro cultured MK and fixed BM tissue. The platelet territory model, in which MK cytoplasm is divided by the membrane system into future platelet territories, is supported by the images by electron microscopy from fixed BM tissue. However, the process of platelet production in vivo remains still enigmatic, because no one could visualize clearly the platelet production from MK in intact BM. Recently, Junt T et al.(1) revealed that MKs routinely release heterogeneous large particles into sinusoids in BM, but their images did not capture the typical long thin proplatelets in sinusoidal lumen using intra-vital microscopy. In this study, we visualized the fine structure of MK protrusions in sinusoidal lumen to reveal the detailed process of platelet production in intact BM of mice skull using intra-vital microscopy. Materials and methods: Two- photon microscope was used to focus on the MK protrusions in sinusoidal lumen of anesthetized green fluorescence protein-transgenic mice to visualize the dynamic platelet production in details. The obtained sequential images were reconstituted as the time-lapse movie, and analyzed in the shape and size of protrusion with the relation to dynamic velocity. To confirm the phenomena captured with the live images, scanning electron microscopy (SEM) was used in fixed BM samples. Results: The time-lapse live images clearly revealed that MK released two types of protrusion into the sinusoidal lumen. One was elliptically-shaped protrusion, followed by typical proplatelets in the sinusoidal lumen (Figure 1). The proplatelet was released as a thin string into sinusoid, but not discoid individual platelet. The other was a thick string, originated from thick protrusions from MK, into sinusoidal lumen (Figure 2). The thick protrusion clearly differed from the typical proplatelet in the size, releasing point, and dynamic movement to the blood flow. Using SEM, we could confirm morphologically two types of platelet production, elliptically-shaped protrusion, followed by typical proplatelets and platelet territory structure in the cytoplasm of thick protrusion. To evaluate the effect of hydrodynamics on proplatelet formation, we evaluated the elongation velocity of proplatelet in sinusoidal lumen by intra-vital imaging. The mean elongating velocity of proplatelet in sinusoidal lumen was 17ƒÊm/min, which was faster than that in static culture condition (0.8ƒÊm/min), implying that sinusoidal flow accelerated the elongation growth of proplatelet. Conclusion: Differed from the previous models based on the live imaging of BM in mice (1), our images captured that the MKs extended and released two types of protrusion into sinusoidal lumens, typical proplatelet without platelet territory and thick protrusion with platelet territory. The structure of thick protrusion with platelet territory suggests a possibility that they have a great advantage for rapid and massive production of circulating platelets in the case of increasing demand for platelets. Proplatelet formation (Figure 1) and thick protrusion (Figure 2) in sinusoid from intra-vital imaging of intact bone marrow in EGFP Tg-mice (megakaryocyte is colored blue and proplatelet is colored green). Disclosures: No relevant conflicts of interest to declare.
A 6-year-old, neutered male Saint Bernard dog was presented with a 1-month history of ataxia, hyp... more A 6-year-old, neutered male Saint Bernard dog was presented with a 1-month history of ataxia, hypermetria and head tilt. High-field magnetic resonance imaging revealed a mass in the cerebellar vermis. During necropsy examination, a cream-coloured irregular area was observed in the cerebellar white matter. Microscopically, the mass comprised a diffuse neoplastic proliferation of spindle cells with oval pleomorphic nuclei in the white and grey matter of the cerebellum and pons and in the subpial area. Neoplastic infiltration was not found in the cerebrum. Immunohistochemistry revealed that the neoplastic cells were positive for vimentin and partially positive for glial fibrillary acidic protein. Based on these findings, the neoplastic lesion was diagnosed as gliomatosis cerebelli, without involvement of the cerebrum.
To study differences in the distribution of proteoglycans and their relationships to collagen fib... more To study differences in the distribution of proteoglycans and their relationships to collagen fibrils in the cornea and sclera, bovine cornea and sclera were examined histochemically using the ruthenium red (RR) staining method. RR-positive granules (30 nm in diameter) were present in association with fine filamentous materials (3 nm in diameter and 30-100 nm in length) in the interfascicular spaces of collagen bundles in both the corneal stroma and sclera. The amount of these materials was smaller in the sclera than in the cornea. The characteristic band-like arrangement of RR-positive granules connected by filamentous materials at intervals of 80-100 nm was found only in the cornea. In enzyme digestion experiments, tissue sections were treated by chondroitinase ABC, AC, and keratanase before RR staining. The RR-positive granules and their associated filamentous materials were darkly stained after chondroitinase AC or keratanase digestion, but displayed markedly lighter staining after chondroitinase ABC digestion. These results indicate that RR-positive granules and filamentous materials contain dermatan sulfate proteoglycan.
Abstract 2300 Introduction: The derivation of blood platelets from bone marrow (BM) MKs has been ... more Abstract 2300 Introduction: The derivation of blood platelets from bone marrow (BM) MKs has been known since 1906, and it evoked long dispute for the process of platelet production between proplatelet model and platelet territory model. The proplatelet model, in which MKs extend long thin protrusion into the sinusoidal lumen and the tip of proplatelet then bud off, is supported by the images from in vitro cultured MK and fixed BM tissue. The platelet territory model, in which MK cytoplasm is divided by the membrane system into future platelet territories, is supported by the images by electron microscopy from fixed BM tissue. However, the process of platelet production in vivo remains still enigmatic, because no one could visualize clearly the platelet production from MK in intact BM. Recently, Junt T et al.(1) revealed that MKs routinely release heterogeneous large particles into sinusoids in BM, but their images did not capture the typical long thin proplatelets in sinusoidal lumen using intra-vital microscopy. In this study, we visualized the fine structure of MK protrusions in sinusoidal lumen to reveal the detailed process of platelet production in intact BM of mice skull using intra-vital microscopy. Materials and methods: Two- photon microscope was used to focus on the MK protrusions in sinusoidal lumen of anesthetized green fluorescence protein-transgenic mice to visualize the dynamic platelet production in details. The obtained sequential images were reconstituted as the time-lapse movie, and analyzed in the shape and size of protrusion with the relation to dynamic velocity. To confirm the phenomena captured with the live images, scanning electron microscopy (SEM) was used in fixed BM samples. Results: The time-lapse live images clearly revealed that MK released two types of protrusion into the sinusoidal lumen. One was elliptically-shaped protrusion, followed by typical proplatelets in the sinusoidal lumen (Figure 1). The proplatelet was released as a thin string into sinusoid, but not discoid individual platelet. The other was a thick string, originated from thick protrusions from MK, into sinusoidal lumen (Figure 2). The thick protrusion clearly differed from the typical proplatelet in the size, releasing point, and dynamic movement to the blood flow. Using SEM, we could confirm morphologically two types of platelet production, elliptically-shaped protrusion, followed by typical proplatelets and platelet territory structure in the cytoplasm of thick protrusion. To evaluate the effect of hydrodynamics on proplatelet formation, we evaluated the elongation velocity of proplatelet in sinusoidal lumen by intra-vital imaging. The mean elongating velocity of proplatelet in sinusoidal lumen was 17ƒÊm/min, which was faster than that in static culture condition (0.8ƒÊm/min), implying that sinusoidal flow accelerated the elongation growth of proplatelet. Conclusion: Differed from the previous models based on the live imaging of BM in mice (1), our images captured that the MKs extended and released two types of protrusion into sinusoidal lumens, typical proplatelet without platelet territory and thick protrusion with platelet territory. The structure of thick protrusion with platelet territory suggests a possibility that they have a great advantage for rapid and massive production of circulating platelets in the case of increasing demand for platelets. Proplatelet formation (Figure 1) and thick protrusion (Figure 2) in sinusoid from intra-vital imaging of intact bone marrow in EGFP Tg-mice (megakaryocyte is colored blue and proplatelet is colored green). Disclosures: No relevant conflicts of interest to declare.
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Papers by Koujiro Tohyama