Natural scrapie: detection of fibrils in extracts from the central nervous system of sheep

Extracts from the cervical spinal cord and from the medulla, thalamus, cerebellum and cerebral cortex of the brains of 10 sheep, histopathologically confirmed as cases of scrapie, were examined by electron microscopy for the presence of scrapie-associated fibrils. Characteristic fibrils were observed in all the extracts except for that from the thalamus of one sheep. No fibrils were found in any extracts from three control sheep. A comparison of these results with a similar study of 22 cases of bovine spongiform encephalopathy (BSE) suggests that in cases of scrapie the area of the brain chosen for the detection of fibrils is less critical than in cases of BSE, in which fibrils are more readily extracted from areas of the brain stem.     

Ultrastructure of canine articular cartilage: comparison of normal and degenerative (osteoarthritic) hip joints.

Normal canine hip cartilage was compared with cartilage from the degenerative lesions found in young dogs with canine hip dysplasia. The upper 0.5 mm of normal cartilage was characterized. Four distinct layers or zones were found: a layer of fine fibrous material covering the surface, a layer (surface layer) of small (32 nm diameter or less) collagen fibrils tightly packed in bundles and oriented parallel to the surface, a layer (upper layer) or less tightly packed collagen fibrils oriented mostly parallel to the surface with about 33% of the fibrils 64 nm or more, and a layer (intermediate layer) of randomly oriented fibrils with more than 50% of the fibrils 64 nm or larger. Fibril density was high in the surface layer and decreased with depth into the cartilage. In a moderately advanced lesion of degenerative cartilage, there was a layer of amorphous material over the surface. The tightly packed surface layer of small fibrils was absent. The surface itself was uneven and fissued. At depths from the surface comparable to the upper and the intermediate layers in normal cartilage, the proportion of large fibrils was less than in normal cartilage. The overall density of fibrils in degenerative cartilage increased with depth into the tissue. Cells flattened parallel to the surface, with relatively large nuclei, were found in the upper layer of normal cartilage. Cells in the intermediate layers were larger and round. The oblong cells of the upper layer of normal cartilage were not found in any layer of degenerative cartilage. Differences between cells in other layers of normal and degenerative cartilages were minimal. A model for the arrangement of chondrocytes and collagen fibrils for normal and degenerative cartilage was proposed. Ultrastructural changes in degenerative cartilage were prominent in the upper 0.5 mm of cartilage. These changes were changes in the number of collagen fibrils/mum-2 and a change from a characteristic pattern of collagen fibril diameters and orientation found in normal tissue.     

The chordae tendineae of the heart in chicken

In this study, the chordae tendineae of 10 adult chickens have been investigated by means of scanning electron microscopy (SEM). It has been observed that the diameter of the collagen fibrils on the left side of the heart was greater than those on the right side. The present study has determined that only perichordal and interchordal ligaments are present in the chicken. It was also observed that axial tendons were surrounded by perichordal membrane. Although no difference was evident among the apex of the chordae, differences were observed between the base and apex of the same chordae.     

Fine Structure of the Bovine Tapetum Fibrosum

The fine structure of the tapetum lucidum of the domestic cow was studied by electron microscopy. In this ungulate the reflective layer is a tapetum fibrosum situated in the choroid and mainly restricted to the superior fundus. The tapetum is composed of a large array of fine extracellular collagen fibrils separated into lamellae by fibrocytes. The collagen fibrils display the normal cross-striations of native collagen, are 0.2 μm in diameter and are arranged in a hexagonal pattern with a center-to-center spacing of about 0.2 μm. The diameter and spacing of these fibrils is consistent with the constructive interference of reflected light. A vascular lamella adjacent to the retinal epithelial layer contains the choriocapillaris and some irregularly arranged collagen fibrils. The tapetum is pierced by blood vessels which supply the choriocapillaris. Over the tapetum the retinal epithelium is non-pigmented while in nontapetal regions this layer is pigmented. The choriocapillaris is not indented into the retinal epithelium as is the case in species with a tapetum cellulosum.     

Scrapie associated fibrils in the diagnosis of scrapie in sheep

Previous research has consistently demonstrated by electron microscopy the presence of scrapie associated fibrils in brain extracts prepared from mice and hamsters with clinical signs of experimental scrapie. In the present study similar fibrils were seen in all the brain extracts prepared from 11 Cheviot or Suffolk sheep with natural or experimental scrapie that had been diagnosed clinically and confirmed neuropathologically. They were not found in the brain extracts of nine sheep that did not have scrapie and which included four that had been injected with infected material but did not develop the disease. The presence of such fibrils can therefore be used as an additional diagnostic criterion for natural scrapie in sheep.     

Calretinin immunoreactive nerve endings in the trachea and bronchi of the rat.

Nerve endings showing calretinin immunoreactivity were examined in the lower respiratory tract of the adult rat. Tree-like nerve endings were immunostained in the tracheal and bronchial smooth muscle layer. The endings that arose from thick nerve fibers and formed corpuscles composed of many arborized nerve terminals. A few of the nerve endings were also observed in the lamina propria of the tracheal mucosa, close to the epithelial layer. Immunoelectron microscopy revealed that the immunoreactive terminals were filled with mitochondria and scattered among the intermuscular collagen fibrils. Schwann cell sheath and collagen fibrils were intercalated between the smooth muscle cells and nerve endings. The calretinin immunoreactive nerve endings observed in the present study seem to be slowly adapting stretch receptors.     

Abnormalities of collagen fibrils in a rabbit with a connective tissue defect similar to Ehlers-Danlos syndrome

A morphometric ultrastructural study was performed to confirm the presence of an abnormality of the collagen fibrils in a rabbit with a connective tissue defect similar to Ehlers-Danlos syndrome. Median fibril diameter and perimeter were not altered but their ranges were significantly increased. As indicated by the median fibril ‘form factor’, fibrils were significantly more irregular in shape; the range of irregularity in shape was also increased. Fibril periodicity was unchanged. The results are discussed in relation to collagen fibril structure and fibril abnormalities in similar diseases in man and other animals.     

不同pH对蛋清溶菌酶形成淀粉样纤维的影响

试验旨在研究不同pH对淀粉样纤维形成的影响。以蛋清溶菌酶（hen egg-white lysozyme,HEWL）为模型蛋白,在不同pH的甘氨酸（50 mmol/L）溶液中（57.0±0.1）℃孵育0~8 d,使其形成不同聚集程度的淀粉样纤维,pH设为2.0、6.5、7.5和8.0;用透射电镜定性观察淀粉样纤维生成状况,采用硫磺素T （ThT）荧光法和刚果红染色法测定淀粉样纤维生长状况,8-苯胺基-1-萘磺酸（ANS）荧光法测定淀粉样纤维疏水性变化,圆二色谱法测定二级结构转化,BeStSel软件预测β-折叠含量。结果显示,透射电镜下观察第8天纤维生长状况发现,pH为2.0时HEWL形成了大量的短杆状淀粉样纤维,同时ThT荧光法结果表明淀粉样纤维生长状况最好（P<0.01）,且生长趋势呈时间依赖性;刚果红染色法显示,在490~510 nm出现红移、540 nm出现特征性肩峰,疏水性显著增强,并且二级结构转化明显,β-折叠含量从天然HEWL蛋白的6.1%增加到37.6%;pH 6.5组仅在透射电镜下观察到极少量的淀粉样纤维,pH 7.5组在视野中未观察到明显的淀粉样纤维,仅观察到大量的球状或杆状蛋白聚集体;pH 8.0组未产生淀粉样纤维。高温条件下,HEWL在强酸性（pH 2.0）条件较易形成淀粉样纤维,且此时疏水性和二级结构转化程度最高,这为解释朊病毒病及其他淀粉样变性疾病的淀粉样纤维形成机制提供了理论基础。     

Bovine spongiform encephalopathy: detection of fibrils in the central nervous system is not affected by autolysis

The effect of autolysis on the electron microscopic detection of the characteristic abnormal fibrils, originally called 'scrapie-associated fibrils', was investigated in four different areas of the central nervous system (CNS) from 10 clinically suspect BSE cattle after post mortem delay and compared with the histopathological diagnosis. The tissues for fibril detection were subjected to controlled incubations to simulate autolysis. Fibril detection in all areas sampled from nine animals in which BSE was confirmed by histopathology was not affected by combined post mortem delays and specific controlled treatments. Detection of fibrils from the cervical spinal cord was no less sensitive than from brain areas. Fibrils were not detected in the one suspect case in which histopathology did not reveal lesions of BSE. The study confirms that fibril detection is of diagnostic value in BSE when post mortem autolysis renders CNS material unsuitable for histopathology.     

The collagen fibrils in fowl medullary bone

Medullary bone from the femurs of laying fowls was examined by electron microscopy, with particular reference to the nature and environment of its collagen fibrils. The collagen fibrils appeared to have a preferred orientation along the long axis of the bone. There were no clear‐cut cyclic changes in the fibril diameters during egg calcification. The tissue was rich in mucopolysaccharides, visualised as strands or sheets connecting the fibrils. Lysis of the fibrils appeared to be associated with osteoclasts rather than osteocytes.     

A connective tissue defect in two rabbits similar to the Ehlers-Danlos syndrome

A connective tissue disease resembling the human Ehlers-Danlos syndrome is reported in two sibling rabbits about four months old. The clinical signs included skin hyperextensibility and fragility and poor wound healing. There were ultrastructural abnormalities in the structure of the collagen fibrils and in their arrangement in bundles of fibres. Affected rabbits may be a useful laboratory animal model for collagen disorders in human beings.     

蛋清溶菌酶淀粉样纤维对人神经母细胞瘤细胞的毒性

本研究旨在探讨一定理化条件下形成的蛋清溶菌酶淀粉样纤维对人神经母细胞瘤细胞（SH-SY5Y）的毒性作用。采用高温（57±0.1）℃、pH 2.0甘氨酸溶液、振荡条件下孵育100 μmol·L^-1的蛋清溶菌酶蛋白，使其聚集成为蛋清溶菌酶淀粉样纤维；透射电子显微镜观察蛋清溶菌酶淀粉样纤维的超微结构，ANS荧光法测定纤维的疏水性，圆二色谱法测定并计算纤维的二级结构含量；将此纤维作用于培养的SH-SY5Y神经细胞，MTT法检测细胞活力，核染色法检测细胞凋亡状况。透射电镜结果显示，蛋清溶菌酶在孵育4 d后形成了呈短杆状超微结构的淀粉样纤维；与天然蛋清溶菌酶蛋白相比，纤维的疏水性和二级结构β折叠含量均显著提高；且1~3 μmol·L^-1纤维作用于SH-SY5Y细胞12、24和48 h均产生了显著的毒性作用（P<0.01），1、2和3 μmol·L^-1纤维作用细胞12 h时的细胞活力分别为75.16%±15.51%、67.54%±12.13%和67.89%±10.26%，作用24 h的细胞活力分别为75.78±13.01%、58.41%±5.55%和61.90%±8.94%，作用48 h的细胞活力分别为71.59%±14.75%、55.65%±5.78%和46.45%±6.23%，细胞活力降低呈现明显的浓度依赖性和时间依赖性，且细胞核染色呈现典型的核凋亡变化。结果提示，蛋清溶菌酶在一定理化条件下可形成具有神经细胞毒性的淀粉样纤维，为解释朊蛋白或其他蛋白质形成淀粉样纤维的机制提供参考。     

Three-dimensional culture of keratinocytes and the formation of basement membrane for canine footpad substitute

A pad equivalent for a dog was prepared as a substitute for the loss of footpad. In addition to the time course of formation on epidermal morphogenesis, we investigated expressions of alpha(6) integrin subunit as adhesive molecule, and laminin and type IV and VII collagens as extracellular matrices of basement membrane components. Epithelium of the pad equivalent was thick enough to be easily confirmed at 5 days at the air-liquid interface, but many creases appeared on it at 7 days, and it shrank at 10 and 14 days. Keratinocytes were increased in 4 to 5 cell layers at 1 day at the air-liquid interface, differentiating into basal cell layer. Granular and corneal cell layers were confirmed until 5 days, and maintained their shape at least until 14 days. Alpha 6 integrin was expressed at almost the same fluorescent intensity as native pad tissue at 1 day at the dermal-epidermal junction. Laminin and type IV collagen were intermittently expressed at 5 and 10 days, respectively, at the dermal-epidermal junction, and at 14 days the fluorescence showed almost the same intensity as native pad tissue. The expression of type VII collagen was discontinuous at 2 days at the dermal-epidermal junction, but remained as it was at 14 days. The present findings suggested that although the formation of anchoring fibrils in basement membrane was incomplete, the pad equivalent in the dog was reconstructed similar to a native pad by epidermal morphogenesis.     

A collagen dysplasia in a greyhound bitch

A 10-month-old greyhound bitch was referred to the Massey University Small Animal Clinic in January 1979, with a history of frequently occurring skin lacerations, especially of the feet and limbs. Clinical findings included multiple skin scars, lacerations and hyperextensibility of the skin.

Investigations showed an increased extensibility index of the skin and a sevenfold reduction in its tensile strength, as compared with that of a normal greyhound. On light microscopy dermal collagen fibres appeared to be slightly decreased in density and more whorled in appearance than that of normal sections. Electron microscopy showed dermal collagen fibrils and bundles which were largely normal, but areas could be found, in the papillary layer of the dermis, where malformed fibrils were prominent, and other areas showed disorganisation of fibrillar packing.

These findings indicated that this bitch had features of a collagen fibrillogenesis disorder similar to some of those described in the various forms of Ehlers-Danlos syndrome in man, and cats and mink. Whether the disease is inherited in the greyhound breed has not yet been determined.     

Comparison of collagen fibre architecture between slow-twitch cranial and fast-twitch caudal parts of broiler M. latissimus dorsi

1. Collagen fibre architectures of perimysium and endomysium in the slow-twitch cranial and fast-twitch caudal parts of broiler M. latissimus dorsi were compared. 2. Type I and III collagens were distributed in both perimysium and endomysium as indicated by their positive immunohistochemical reactions to polyclonal antibodies. 3. Cells invested by endomysium with no myofibres were larger in the cranial part because of the presence of larger slow-twitch myofibres. The honeycomb structure of endomysium was divided into several parts by thick perimysium. 4. The thick perimysial collagen fibres with parallel fibrils, which were interconnected by the loose reticular fibrils and thin fibres, were more numerous and thicker in the cranial part than the caudal. 5. Thick endomysial sidewall of cells in the cranial part was composed of a rougher reticulum of slightly thicker collagen fibrils compared with the thin sidewall in the caudal part. 6. These results indicated that both perimysial constitutions of collagen fibres and endomysial collagen fibrils had attained much larger growth in the slow-twitch cranial part than the fast-twitch caudal in broiler latissimus dorsi muscle.     

Depiction of the structure of the vitreous body in horses without ocular diseases and in horses with equine recurrent uveitis (ERU) using transmission electron microscopy

Neither the ultrastructure of the vitreous body from horses without ocular diseases, nor the pathomorphological changes in the vitreous body associated with equine recurrent uveitis (ERU) have been described. However, the vitreous body plays an important role in the pathogenesis of ERU. Ten vitreous body samples obtained from 5 horses without ocular disease, and 38 vitreous body samples from horses with ERU (collected during vitrectomy) were examined by transmission electron microscopy. The vitreous body samples of horses without ocular diseases were characterized by a loose network of unbranched fibrils 10-12 nm in width. In the vitreous body samples of horses with ERU numerous dense bundles of fibrils, mononuclear inflammatory cells and necrotic cells represent the destruction of the vitreous fibrillar network. In this study, equine vitreous body ultrastructure was described for the first time. Thus, demonstrating ultramorphologically, the clinically apparent changes of the vitreous body associated with ERU.     

Amyloid angiopathy with cerebral hemorrhage and senile plaque in aged dogs

Amyloid angiopathy with cerebral hemorrhage and senile plaques was found in the brain of aged dogs. In all 9 cases examined, 13 to 19 years old, 6 males and 3 females, amyloid deposits were observed mostly in the wall of cerebral arterioles and capillaries showed hyaline degeneration. The accumulation of amyloid fibrils measuring about 10 nm in diameter was seen in the cerebral vessel wall by electron microscopy. The cerebral hemorrhage was observed in 6 of 9 dogs and 2 of them showed massive hemorrhage. The hemorrhagic foci were sometimes closely contact with the vessels involved in amyloid angiopathy. In addition, senile plaques being classified into 2 types were found in the cerebral cortex of 3 dogs. The first type was characterized by the accumulation of degenerative neurites and often contained granular argyrophilic material. The second type had a well-defined amyloid core with neuritic halo. Amyloid deposits were also found in or around intestinal vessel walls of 3 dogs. The amyloid deposited in the cerebral vessels, senile plaques and intestinal vessels showed characteristic green birefringence under the polarized light even after potassium permanganate treatment.     

Vitiligo in a Quarter Horse Filly: Clinicopathologic, Ultrastructural, and Nutritional Study

Vitiligo is an autoimmune disorder characterized clinically by depigmentation of the skin—both epidermis and hairs—caused by destruction of melanocytes. Vitiliginous skin shows a T-lymphocyte inflammatory infiltrate, proliferation of Langerhans cells, epidermal vacuoles, and degenerative changes in the cutaneous nerve fibrils. Increased autoantibodies against melanin, tyrosinase, and various tissues are frequently present in vitiligo patients. Vitiligo is often associated with systemic abnormalities or nutritional deficiencies. A 2-year-old Quarter Horse filly developed facial vitiligo with depigmented areas that was strikingly similar to human vitiligo. Several innocuous small biopsies provided cutaneous specimens that were processed for light and electron microscopic studies. Many of the clinical and microscopic changes observed in human vitiligo were present in this equine patient. Marginal anemia was detected. Strengthening of the filly's nutritional and feeding conditions led to rapid and complete repigmentation. Equine practitioners as well as horse owners should be aware that vitiligo may not be simply a cosmetic problem; thus, a complete evaluation of horses affected by this condition should be performed.     

Ultrastructure features of camel cornea--collagen fibril and proteoglycans

Introduction The uniform distribution of collagen fibrils and proteoglycans maintain the transparency of normal cornea. We describe the ultrastructural features of camel cornea including collagen fibrils and proteoglycans (PGs). Methods Camel corneas (of 6‐, 8‐, and 10‐month‐old animals) were fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer and processed for electron microscopy. The ‘AnalySIS LS Professional’ program was used to analyze the collagen fibril diameter. Results The camel cornea consists of four layers: the epithelium (227 μm), stroma (388 μm), Descemet’s membrane (DM), and endothelium. The epithelium constituted 36% of the camel cornea, whereas corneal stroma constituted 62% of the corneal thickness (629 μm). The PGs in the posterior stroma were significantly larger in number and size compared with the anterior and middle stroma. The collagen fibril diameter was 25 nm and interfibrillar spacing 40 nm. Fibrillar structures are present throughout the DM. Conclusion The structure of the camel cornea is very different from human and other animals. The unique structure of the cornea might be an adaptation to help the camel to survive in a hot and dry climate. The camel cornea may also be a good model to study the effect of hot and dry climates on the cornea.     

Ehlers-Danlos-like syndrome in 2 dogs: clinical,histologic, and ultrastructural findings

Background: Ehlers-Danlos syndrome comprises a group of rare inherited connective tissue diseases characterized by skin hyperextensibility, joint laxity, skin and vessel fragility, and poor wound healing.
Objective: The purpose of this report was to describe the clinical, histologic, and ultrastructural findings in 2 dogs with collagenopathies consistent with Ehlers-Danlos syndrome.
Methods: Two dogs were examined clinically; skin extensibility index was calculated. Skin biopsies obtained from the dorsum were examined by light and electron microscopy.
Results: Both dogs had clinical signs of skin hyperextensibility and fragility, lower skin elasticity, vessel fragility, and poor wound healing. One dog had a hip dislocation, and the other had bilateral medial patellar luxation (grade II), subcutaneous hematomas produced by minimal trauma, and generalized periodontitis. Histologic and ultrastructural examination confirmed abnormalities in the structure and arrangement of collagen fibrils. Fibroblasts were characterized by variable dilatation of the rough endoplasmic reticulum, and anomalous elastic fibers (elaunin fibers) were present in the dermis.
Conclusion: Although the primary defects underlying collagenopathies in animals are still unknown, analysis of the ultrastructural changes in collagen fibrils and clinical findings could facilitate better characterization of these disorders in dogs.