Histopatological parameters of the spinal cord in different phases of experimental autoimmune encephalomyelitis. A mouse model of multiple sclerosis examined by classical stainings combined with immunohistochemistry.

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE) are characterized by three main histopathological parameters: inflammation, demyelination and axonal damage. In this study, these parameters were assessed in spinal cords of mice in the successive phases of EAE by quantitative histology and immunohistochemistry. The number of inflammatory lesions, the intensity of inflammation and expression of CD45 corresponded with the severity of clinical symptoms: they increased from the onset phase to the peak phase of the disease and subsided in the chronic phase. Demyelination increased in the peak phase and did not change in the chronic phase of EAE, although axonal damage gradually increased from the onset phase to the chronic phase, suggesting compensatory hypermyelination in that phase. The markers of myelin and axonal injury: myelin basic protein (MBP) and beta amyloid precursor protein (ß-APP) showed changes (decrease and increase, respectively) of expression parallel to changes in demyelination and axonal damage. Results of this study indicate that although inflammation intensity subsides in the chronic phase of EAE, the neurodestructive processes: demyelination and axonal damage continue in that phase.

Changes in stiffness of the optic nerve and involvement of neurofilament light chains in the course of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis.

Multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), are often accompanied by optic neuritis associated with neurofilament disruption. In this study, the stiffness of the optic nerve was investigated by atomic force microscopy (AFM) in mice with induced EAE in the successive phases of the disease: onset, peak, and chronic. AFM results were compared with the intensity of the main pathological processes in the optic nerve: inflammation, demyelination, and axonal loss, as well as with the density of astrocytes, assessed by quantitative histology and immunohistochemistry. Optic nerve tissue and serum levels of neurofilament light chain protein (NEFL) were also examined by immunostaining and ELISA, respectively. The stiffness of the optic nerve in EAE mice was lower than that in control and naïve animals. It increased in the onset and peak phases and sharply decreased in the chronic phase. Serum NEFL level showed similar dynamics, while tissue NEFL level decreased in the onset and peak phases, indicating a leak of NEFL from the optic nerve to body fluids. Inflammation and demyelination gradually increased to reach the maximum in the peak phase of EAE, and inflammation slightly declined in the chronic phase, while demyelination did not. The axonal loss also gradually increased and had the highest level in the chronic phase. Among these processes, demyelination and especially axonal loss most effectively decrease the stiffness of the optic nerve. NEFL level in serum can be regarded as an early indicator of EAE, as it rapidly grows in the onset phase of the disease.

Biomechanical changes in the liver tissue induced by a mouse model of multiple sclerosis (EAE) and the effect of anti-VLA-4 mAb treatment.

Experimental autoimmune encephalomyelitis (EAE) is a mouse model of demyelinating diseases, such as multiple sclerosis (MS). MS can be accompanied by autoimmune hepatitis. In this study, nanomechanical, biorheological and histological examinations were carried out by atomic force microscopy (AFM), rheology, and immunofluorescence microscopy to investigate changes in the liver tissue of EAE mice and the effect of natalizumab, a monoclonal antibody against α4-integrin (VLA-4) cell adhesion molecule, used in MS therapy. Liver samples collected from EAE mice in three successive phases of the disease showed inflammatory changes manifested by leukocyte infiltrations and elevated levels of proinflammatory cytokine IL-1ß. Liver stiffness and viscoelasticity increased in the onset phase of EAE, decreased in the peak phase and increased again in the chronic phase to reach the highest values. These changes were not associated with inflammation parameters which increased in the peak phase and decreased to the lowest values in the chronic phase. Moreover, anti-VLA treatment, which reduced the inflammation parameters, had an ambiguous effect on stiffness and viscoelasticity: it increased them in the peak phase but decreased in the chronic phase. The observed discrepancies can result from a complex network of interactions between inflammation and fibrosis, as well as between liver cells and the extracellular matrix influencing the biomechanical properties of the liver tissue.

Effect of natalizumab treatment on metalloproteinases and their inhibitors in a mouse model of multiple sclerosis.

Matrix metalloproteinases (MMPs) regulated by their tissue inhibitors (TIMPs) play a significant role in the pathogenesis of multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis (EAE), as they degrade extracellular matrix including vascular basal laminae and by damaging blood-brain barrier (BBB) facilitate transmigration of immune cells into the central nervous system. MMPs are also involved in destruction of myelin sheaths, leading to axonal and neuronal loss. The aim of the present study was to assess whether natalizumab, a transmigration-inhibiting monoclonal antibody against α4ß1 integrin, influences expression of MMPs and TIMPs in the central nervous system of mice with EAE. MMP-2 and MMP-9, their respective inhibitors TIMP-2 and TIMP-1 and laminin were assessed by quantitative immunohistochemistry in the spinal cord cryosections of C57BL/6 mice with EAE in the successive phases of the disease (onset, peak and chronic). The percentage of immunopositive areas were calculated in sections encompassing the whole spinal cord cross-sectional area occupied by the gray and white matter. Results obtained in animals administered with 5 mg/kg natalizumab were compared with those collected from control mice receiving 5 mg/kg IgG. Both studied MMPs and both TIMPs were upregulated in control EAE mice. Natalizumab treatment significantly reduced expression of MMPs and increased expression of TIMPs in the peak and chronic phases of the disease. This effect was accompanied by inhibition of laminin degradation in the vascular basal laminae and reduction of inflammatory infiltration. Results of this study demonstrate that in addition to its well known anti-integrin activity counteracting transmigration of immune cells into the central nervous system, natalizumab strengthens this effect by its probably indirect influence on MMPs and TIMPs leading to protection of blood-brain barrier integrity.

Changes in spinal cord stiffness in the course of experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis.

Experimental autoimmune encephalomyelitis (EAE) is a commonly used mouse model of multiple sclerosis, a chronic inflammatory disease of the central nervous system (CNS) characterized by demyelination leading to brain and spinal cord malfunctions. We postulate that not only biological but also biomechanical properties play an important role in impairements of CNS function. Atomic force microscopy (AFM) was applied to investigate mechanical properties of spinal cords collected from EAE mice in preonset, onset, peak, and chronic disease phases. Biomechanical changes were compared with histopathological alterations observed in the successive phases. The deformability of gray matter did not change, while rigidity of white matter increased during the onset phase, remained at the same level in the peak phase and decreased in the chronic phase. Inflammatory infiltration and laminin content accompanied the tissue rigidity increase, whereas demyelination and axonal damage showed an opposite effect. The increase in white matter rigidity can be regarded as an early signature of EAE.

Elevated serum osteoprotegerin is associated with decreased osteoclastic differentiation in stenotic aortic valves.

Calcific aortic valve stenosis (CAVS) is an actively regulated process that involves mechanisms of bone development, including the receptor activator of nuclear factor κB, its ligand, and osteoprotegerin (RANK/RANKL/OPG) regulatory system. The aim of this study was to investigate whether the levels of circulating OPG and RANKL can be correlated with some histopathological features of the stenotic valves. Serum levels of osteoprotegerin (OPG) and soluble RANKL (sRANKL) were assessed in 27 patients with CAVS prior to valve replacement surgery and in 12 control subjects. The removed valves were examined macroscopically and microscopically. Valve sections were stained with hematoxylin and eosin for general morphology, with Oil Red O for lipids and immunostained with antibodies against markers visualizing osteoclastic cells (tartrate-resistant acid phosphatase, TRAP), macrophages (CD68) and blood vessels (CD34). Patients with CAVS had elevated levels of OPG as compared to the control group (p=0.005). Within the CAVS group, patients with osteoclastic TRAP-positive cells in their valves had significantly lower serum levels of OPG (p=0.009) and lipid content (p=0.03) than those without such cells. Moreover, osteogenic metaplasia was observed exclusively in the valves containing TRAP-positive cells. Results of this study suggest that the circulating OPG can influence the processes occurring in the calcifying valves by inhibiting osteoclastic differentiation of cells involved in calcification and by preventing osteogenic metaplasia.

Vasodilatory effect and endothelial integrity in papaverine- and milrinone-treated human radial arteries.

Prevention of the vasospasm is an important aspect of coronary artery bypass grafting (CABG) with the use of radial artery (RA) as the conduit. We compared the effect of two phosphodiesterase inhibitors papaverine and milrinone on vasodilation and endothelial integrity of human RA segments harvested from 20 CABG patients. Vasodilatory effect of the drugs were assessed by organ bath technique in RA rings precontracted with KCl and phenylephrine. Endothelial integrity was evaluated by CD34 immunofluorescence in frozen sections. Vasorelaxation induced by papaverine was significantly greater as compared to that induced by milrinone (90.47% ± 10.16% vs. 78.98% ± 19.56%, p<0.05). Similarly, pretreament with papaverine more strongly inhibited the contractile response of RA rings to KCl (6.0 ± 8.0 mN vs. 26.7 ± 21.5 mN, p<0.001). Papaverine was also superior to milrinone in the preservation of endothelial integrity (75.3% ± 12.9% vs. 51.8% ± 18.0%, p<0.02). In conclusion, papaverine seems to be more suitable than milrinone for prevention of vasospasm in radial artery conduits used for CABG.

Effect of nebivolol treatment on atherosclerotic plaque components in apoE-knockout mice.

Nebivolol, a third generation beta1-blocker was previously found to reduce the size of atherosclerotic lesions. The aim of this study was to assess the effect of orally administered nevibolol on the components of the atherosclerotic plaque in apoE-deficient mice. The quantitative evaluation of cross-sectioned plaques stained by histological and immunohistochemical techniques revealed that treatment with nebivolol (2.0 µol per kg b.w.) for 4 months caused a decrease in the necrotic core area (by 46%, p=0.03), density of CD68+ macrophages (by 41%, p=0.008) and CD3+ lymphocytes (by 16%, p=0.03), collagen content (by 49%, p=0.008) and the activity area of metalloproteinases (by 48%, p=0.008), as well as an increase in the smooth muscle content of the fibromuscular cap (by 46%, p=0.008). These effects suggest that nebivolol suppresses the inflammatory/immune processes in the plaque and enhances its stability.

Effect of cultured autologous oral keratinocyte suspension in fibrin glue on oral wound healing in rabbits.

The effect of cultured autologous oral keratinocyte suspension in fibrin glue on the healing of surgically produced oral mucosal wounds was assessed in the rabbit model. Using the light microscope and a digital image analysis system, the epithelization parameters (marginal epithelization and percentage of wound re-epithelization) were measured in haematoxylin-eosin stained sections of the wound area and compared with those of wounds treated with fibrin glue alone and untreated ones. The epithelization was significantly higher in keratinocytes plus fibrin glue-treated wounds on postoperative days 3 and 7. No significant differences were observed on postoperative day 1, when the healing process had just begun, and on postoperative day 14, when re-epithelization was completed or nearly completed in all groups. The inflammatory infiltration of the wounded mucosa was weakest in keratinocyte-treated wounds and strongest in untreated wounds. In conclusion, suspension of cultured autologous oral keratinocytes in fibrin glue significantly accelerates oral wound healing in the rabbit model and could be beneficial in the treatment of oral wounds in patients.

Development of galanin-containing nerve fibres in rat tibia.

Galanin exerts tonic inhibition of nociceptive input to the central nervous system. Recently, this peptide was demonstrated in several neuronal and non-neuronal structures in bones and joints. In this study, the time of appearance and topographic localization of galanin-containing nerve fibres in bone were studied in rats from gestational day 16 (GD16) to postnatal day 21 (PD21). The tibia was chosen as a model of developing long bone and indirect immunofluorescence combined with confocal laser scanning microscopy was used to identify galanin-immunoreactive (GAL-IR) nerve fibres. The earliest, sparse GAL-IR fibres were observed on GD21 in the perichondrium of both epiphyses and in the periosteum of the diaphysis. From PD1 onwards, GAL-IR fibres were also seen in the bone marrow cavity and in the region of the inter-condylar eminence of the knee joint. Intramedullary GAL-IR fibres in proximal and distal metaphyses appeared around PD1. Some of them accompanied blood vessels, although free fibres were also seen. GAL-IR fibres located in the cartilage canals of both epiphyses were observed from PD7, in the secondary ossification centres from PD10 and in the bone marrow of both epiphyses from PD14. The time course and localization of galanin-containing nerve fibres resemble the development of substance P- and CGRP-expressing nerve fibres, thus suggesting their sensory origin.

Arterial supply and venous drainage of the choroid plexus of the human lateral ventricle in the prenatal period as revealed by vascular corrosion casts and SEM.

The topography of the arterial supply and venous drainage was visualised by corrosion casting and scanning electron microscopy in the human foetal (20 weeks) choroid plexus of the lateral ventricle. Although secondary villi were not yet present at that developmental stage, the topography of the large arteries and veins almost fully corresponded to that described in adult individuals. The only major difference observed was a lack of the typical tortuosity of the lateral branch of the anterior choroidal artery and of the superior choroidal vein, which probably develops during further expansion of the vascular system associated with the formation of secondary villi.

The microvascular architecture of the choroid plexus in fetal human brain lateral ventricle: a scanning electron microscopy study of corrosion casts.

The microvascular architecture of developing lateral ventricle choroid plexus was investigated by corrosion casting and scanning electron microscopy in human fetuses aged 20 gestational weeks. The areas with different microvascular patterns corresponded to the particular parts of the mature plexus: anterior part, glomus, posterior part, the villous fringe and the free margin. In the posterior part, densely packed parallel arterioles and venules were surrounded by sheath-like capillary networks. Other areas contained compact capillary plexuses of the primary villi: the most prominent, protruding basket- and leaf-shaped plexuses were observed in the villous fringe, whilst less numerous and smaller plexuses occurred in the anterior part and glomus. The capillaries of the plexuses had a large diameter and sinusoidal dilations, and showed the presence of occasional short, blind sprouts indicative of angiogenesis. Short anastomoses between arterioles supplying the plexuses and venules draining them were only rarely observed. In the upper area of the choroid plexus, the superior choroidal vein was surrounded by a capillary network forming small, glomerular or rosette-shapes plexuses. The free margin of the choroid plexus was characterized by flat, multiple, arcade-like capillary loops. The general vascular architecture of the human choroid plexus at 20 gestational weeks seems to be similar to that of postnatal/mature plexus, still lacking, however, the complex vascular plexuses of the secondary villi.

The structure of acquired aural cholesteatoma as revealed by scanning electron microscopy.

The structural features of cells, their surfaces and the extracellular matrix were investigated in acquired aural cholesteatoma. Cholesteatomas surgically removed from 30 patients were examined by a scanning electron microscope (SEM). The predominant part of a cholesteatoma was composed of stratified squamous epithelium, showing extensive chaotic desquamation. The surface sculpture of the keratinocytes and corneocytes varied from parallel ridges, irregular microplicae and mirovilli, to flat grooves and pits and a completely smooth surface. Sheetlike lamellar structures, probably representing an intercellular lipid-forming permeability barrier, were also observed. Small crystals located in the perimatrix were observed in one case. According to the SEM observations, cholesteatoma epithelium is characterised by abnormal and uncoordinated keratinisation, with a predominance of the advanced stages of the process.

Surface structures of aural polyps: a scanning electron microscopic study.

Surfaces of aural polyps collected from 30 patients were examined by scanning electron microscopy. In the polyps not associated with cholesteatoma, the epithelial lining showed individually variable metaplasia towards cuboidal 'cobblestone'-type and squamous epithelium covered with microvilli of various shapes and sizes. Squamous epithelium was present on the surface of all polyps with underlying cholesteatoma, with superficial cells possessing elongated microvilli, microplicae of different sizes, grooves and pits. Such surface structures reflect different stages of the keratinization process that seems to be characteristic for the epithelial lining of polyps with underlying cholesteatoma. Incomplete epithelium accompanied by granulation tissue was found in several polyps; in two cholesteatoma-associated polyps plate-like cholesterol crystals were observed.

Vascular architecture of normal human urinary bladder and its remodeling in cancer, as revealed by corrosion casting.

The vasculature of normal human urinary bladder wall and its tumors were studied using corrosion casting and scanning electron microscopy. In the normal wall, a regular organization of highly tortuous horizontal and vertical vessels allows accommodation of the vascular system to spatial changes resulting from the filling/voiding cycle. The vasculature of tumors is a result of remodeling of preexisting wall vessels associated with gradual growth of the neoplastic tissue. The shape and arrangement of blood vessels in different regions of the tumor seem to reflect a variable dynamics of tumor development and possible influence of various angiogenesis-promoting growth factors.

Vascular system of the human spinal cord in the prenatal period: a dye injection and corrosion casting study.

The vascularization of the spinal cord was investigated in 50 human fetuses aged from 10 to 28 gestational weeks using dye injection methods and corrosion casting accompanied by scanning electron microscopy. In the investigated period of fetal development, the general vascular architecture of the spinal cord, corresponding to that described postnatally, seemed to be already established. The observed changes included: (1) remodeling of the supplying (extrinsic) arterial branches, (2) transformation of the posterior anastomotic chain into two distinct posterior spinal arteries, and (3) development of the capillary networks in the gray and white matter. The remodeling of the radicular arteries supplying the spinal cord was accompanied by a decrease in their number and transition from regular to irregular distribution (appearance of intersegmental differences in their frequency). The anterior spinal artery and regular array of the central arteries were already present in the youngest fetuses examined, but the final remodeling of the posterior anastomotic chain into two posterior spinal arteries occurred between 15th and 20th week of fetal life indicating that the vascularization of the anterior region of the spinal cord in the investigated period of fetal life was more advanced as compared with that of the posterior region. The capillary network of the gray matter in the youngest fetuses had the form of discrete glomerular plexuses supplied by groups of central arteries and mainly vascularizing the anterior horns. Successively, the plexuses fused to form a continuous system along the anterior columns and the system expanded to fully vascularize the posterior horns. The white matter in the earlier fetal period seemed to be partially avascular, later the density of capillaries vascularizing those areas was still much lower than in the gray matter. The veins showed considerably greater variability than the arteries, as far as their topography and distribution was concerned. High tortuosity characterized the superficial veins, especially in the younger fetuses, although the degree of tortuosity differed even between individual fetuses. Only anterior spinal and central arteries were usually accompanied by their venous counterparts, the other veins seemed to have no regular topographical relations with the arteries.

Morphological and clinical characteristics of antrochoanal polyps: comparison with chronic inflammation-associated polyps of the maxillary sinus.

OBJECTIVES: to compare morphological and clinical features of antrochoanal polyps and chronic inflammation-associated polyps of the maxillary sinus. STUDY DESIGN: histological and scanning electron microscopic examination of ten antrochoanal polyps and ten chronic inflammation-associated polyps of the maxillary sinus; comparison of clinical data in both groups of patients. METHODS: following surgical removal, the polyps were halved, the halves being processed for routine light microscopy (formalin fixation, paraffin embedding, HE staining) and scanning electron microscopy (formaldehyde/glutaraldehyde fixation, critical point drying, gold coating), respectively. Clinical data were retrospectively reviewed, tabulated and compared. RESULTS: the antrochoanal polyps differed from chronic inflammation-associated polyps of the maxillary sinus only in a few minor features; slightly longer duration of the process, lower incidence of maxillary ostial obstruction, higher incidence of frequent headaches, persistent nasal obstruction, presence of cysts in the polyp stroma, thickened basement membrane, lower incidence of squamous cell metaplasia, and higher proportion of migratory cells in nasal smears. In two cases, allergy was diagnosed but it seemed not to influence the polyps, which did not show morphological features typical of allergy-associated (eosinophilic) polyps. CONCLUSIONS: In spite of minor differences, antrochoanal polyps can be regarded as chronic inflammation-associated polyps with cystic origin and peculiar localization.

Maxillary sinus polyps secondary to dental extraction.

Polyps of the maxillary sinus were obtained from six patients who had reported upper tooth extraction with oroantral perforation prior to the development of symptoms, from 11 patients with chronic sinusitis, and from 12 patients with allergy. Histopathological features, scanning electron microscopy of the polyp epithelium and clinical data were compared in those groups of patients. The post-traumatic polyps differed from those of other aetiologies by showing the presence of granulomas, less numerous inflammatory cells with very few eosinophils, nearly normal surface epithelium (smaller surface area occupied by nonciliated epithelium, absence of epithelial squamous cells, normal frequence of goblet cells), rapid appearance of symptoms, and shorter duration of the disease. It seems that the specific characteristics of the injury-induced polyps results from a different mechanism of their formation, involving primarily abnormal mucosal repair and to a lesser extent an inflammatory process.

Immunocytochemical investigation of catalase and peroxisomal lipid beta-oxidation enzymes in human hepatocellular tumors and liver cirrhosis.

A significant reduction of catalase activity, a peroxisomal marker enzyme, occurs in human hepatic neoplasias, but no information is available on other peroxisomal proteins. We have studied by means of immunohistochemistry four specific proteins of peroxisomes (catalase and three enzymes of lipid beta-oxidation) in human hepatocellular tumors of various differentiation grades from adenoma to anaplastic carcinoma. In all tumors, except the adenomas, the tumor cells contained fewer peroxisomes than extrafocal hepatocytes and the reduction of antigenic sites in the tumor types generally correlated with the degree of tumor dedifferentiation as assessed by classical histopathological criteria. Two poorly differentiated tumors had no detectable peroxisomes at all. There were no major differences in the intensities of the immunocytochemical staining for all four studied peroxisomal antigens in different tumors, suggesting that the neoplastic transformation affects the biogenesis of the entire organelle and not merely the individual peroxisomal enzyme proteins. Some tumors exhibited a distinct peripheral distribution of peroxisomes. In cases with associated liver cirrhosis, the hepatocytes in the adjacent liver showed marked peroxisome proliferation, forming large perinuclear aggregates, occupying occasionally the entire cytoplasm. Taken together, our observations indicate that peroxisomes are significantly altered in both hepatocellular tumors and liver cirrhosis and, thus, could be responsible for some of the metabolic derangements observed in those disease processes.

Architecture of blood vessels in human fetal gastric corpus: a corrosion casting study.

Vascular architecture of the gastric corpus was investigated in 16-24 wk human fetuses using a corrosion casting technique and the scanning electron microscopy. The general distribution of blood vessels seen in adults has already been established in the fetus, with three major vascular plexuses located in the serosa, submucosa and mucosa. The serosal plexus, supplied and drained by large extramural vessels, contained anastomosing, arcade-like arrays of arteries and veins with their branches piercing the muscularis and communicating with the compact submucosal plexus. Vertical arterioles and capillaries were sent by submucosal arteries to supply a very dense capillary plexus which surrounded the gastric pits and consisted of wide, sinusoidal vessels showing morphological manifestations of angiogenesis by intussusceptive growth. The plexus was drained by vertical venules emptying into submucosal veins. In contrast to the richly vascularized upper half of the mucosa, the lower half showed a relative paucity of blood vessels, probably due to the thinness of the fetal mucosa allowing an effective diffusion of oxygen and nutrients from the upper half. Neither arteriovenous anastomoses, nor end-arteries were found in the fetal stomach. Results of this study support one of the two existing models of mucosal vascularization in the human stomach: i.e. the model postulating the presence of short and long arterioles and two distinct, albeit interconnected capillary networks in the upper and lower zones of the mucosa respectively. In human fetuses, the latter network is absent; it probably develops by remodelling of the preexisting vertical capillaries in the last phase of pregnancy, prior to the onset of gastric gland function.