Refinement and revalidation of the Equine Ophthalmic Pain Scale: R-EOPS a new scale for ocular pain assessment in horses.

This study addresses the refinement and revalidation of a composite pain scale that focuses on equine facial expressions and behavioural indicators as exhibitions of ophthalmic pain. This scale included only Behavioural and Facial and Ocular expression indicators and, compared to the first version of Equine Ophthalmic Pain Scale (EOPS), item descriptors and related ratings were changed. Thirteen horses with ocular diseases that required medical or surgical treatment were enroled (group P). In each animal, the refined EOPS (R-EOPS) was applied prior to any treatment (T0) and one week later (T7). The R-EOPS was applied twice, 7 days apart, to 16 healthy control horses (group C). Two 30-second videos were recorded each time to allow the retrospective analysis by eight observers. Inter-observer reliability of items was moderate or substantial (Krippendorff's alpha, Kα>0.40) while their intra-observer reliability was substantial or almost perfect for most items (Kα ≥0.61). Both inter- and intra-observer reliability of Total Score (TS) were however excellent (Intraclass Correlation Coefficients, ICC>0.75). The TS also showed good reproducibility (Kendall coefficient=0.786, ICC=0.684) and high consistency of its items (Cronbach's α=0.847). The comparison between groups as well as the sensitivity and specificity values supported the validity of the R-EOPS. In particular, for each extra point added to the TS, the risk of the horse having pain increased by more than two times (Odds Ratio=2.079, 95%CI=1.542-2.804; P<0.001). The Receiver Operating Characteristic analysis identified 6 as the threshold value of R-EOPS for discriminating horses with ocular pathology (sensitivity=83%, specificity=100%). This scale may be an effective tool for reliably assessing the pain level in horses with ophthalmic diseases and potentially guiding pain management although it still requires large-scale application and external validation.

Development and preliminary validation of a pain scale for ophthalmic pain in horses: The Equine Ophthalmic Pain Scale (EOPS).

The purpose of this study was to describe the development and preliminary validation of a composite pain scale, called the Equine Ophthalmic Pain Scale (EOPS), to assess ocular pain in horses. Indicators associated with ocular pain were selected and classified as behavioural, physiological or ocular expressions. Eight horses diagnosed with ocular or adnexa diseases that required medical or surgical treatment were enrolled in the study (group P). The developed EOPS was applied at the baseline (T0) and 1 week later (T7). Moreover, the EOPS was applied twice, 1 week apart, to 15 healthy control horses (group C). Videos of 60-80 s duration of all assessments were retrospectively analysed by seven masked observers, who scored items included in the behavioural and ocular expression categories of the EOPS. The inter- and intra-observer reliability was excellent (intraclass correlation coefficients ≥0.75) for most of the scored items. Cronbach's alpha (0.76) indicated that the EOPS had good internal consistency. The total score (TS), calculated as the sum of all scores, differed between groups C and P at T0 (P < 0.001) and reduced after medical/surgical treatment in group P (P = 0.017), indicating the responsiveness of the EOPS. Moreover, the area under the curve (AUC=0.918, 95% confidence interval = 0.815-1.000; P < 0.001) indicated that the EOPS was very accurate for distinguishing healthy from pathological animals. Sensitivity and specificity of EOPS to identify horses with ocular pathology (at the optimal cut-off, i.e. TS ≥ 7) were 81.3% and 100.0%, respectively. However, 'overall behaviour', 'position inside the box', 'ear movements' and 'head position' items as well as physiological parameters, showed sub-optimal reliability, consistency and/or item-total correlation, suggesting that there is still room to improve this composite scale.

Carotenoids co-localize with hydroxyapatite, cholesterol, and other lipids in calcified stenotic aortic valves. Ex vivo Raman maps compared to histological patterns.

Unlike its application for atherosclerotic plaque analysis, Raman microspectroscopy was sporadically used to check the sole nature of bioapatite deposits in stenotic aortic valves, neglecting the involvement of accumulated lipids/lipoproteins in the calcific process. Here, Raman microspectroscopy was employed for examination of stenotic aortic valve leaflets to add information on nature and distribution of accumulated lipids and their correlation with mineralization in the light of its potential precocious diagnostic use. Cryosections from surgically explanted stenotic aortic valves (n=4) were studied matching Raman maps against specific histological patterns. Raman maps revealed the presence of phospholipids/triglycerides and cholesterol, which showed spatial overlapping with one another and Raman-identified hydroxyapatite. Moreover, the Raman patterns correlated with those displayed by both von-Kossa-calcium- and Nile-blue-stained serial cryosections. Raman analysis also provided the first identification of carotenoids, which co-localized with the identified lipid moieties. Additional fit concerned the distribution of collagen and elastin. The good correlation of Raman maps with high-affinity staining patterns proved that Raman microspectroscopy is a reliable tool in evaluating calcification degree, alteration/displacement of extracellular matrix components, and accumulation rate of different lipid forms in calcified heart valves. In addition, the novel identification of carotenoids supports the concept that valve stenosis is an atherosclerosis-like valve lesion, consistently with their previous Raman microspectroscopical identification inside atherosclerotic plaques.

Immunolocalization of interleukin-1 receptor antagonist in healthy and infarcted myocardium.

Ischemic heart disease is a widespread cause of death. During infarction, myocardial injury is mediated by release of several pro-inflammatory cytokines including multifunctional interleukin-1 (IL-1). In various tissues, IL-1-mediated deleterious effects are known to be attenuated via the over-expression of natural anti-inflammatory cytokine IL-1 receptor antagonist (IL-1ra). In the present investigation, IL-1ra distribution in healthy and infarcted myocardium was studied by light and electron microscopy. After immunostaining, weak positivity resulted for cardiomyocytes in normal myocardium and, at higher degrees, in infarction border areas and ischemic ones. In ischemic areas, additional reactivity was displayed by the extracellular matrix and intravascular plasma. Immunogold labelling provided further details on intracytoplasmatic and extracellular distribution; in particular, noticeable gold particle distribution appeared on intercalated discs in normal and hypertrophic cardiomyocytes, as well as on thickened Z-lines for these latter. The present results suggest that cardiomyocytes represent a major source of IL-1ra in vivo, even though additional contribution by blood derived IL-1ra is to be taken in account in ischemic areas. In addition, ischemia-associated intracytoplasmic IL-1ra increase and its additional presence in the extracellular matrix is consistent with the concept that this cytokine plays a cardioprotective role at different levels and by distinct mechanisms.

Immunohistochemical profile of some neurotransmitters and neurotrophins in the seminiferous tubules of rats treated by lonidamine.

Lonidamine (LND) or [1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid] is an anticancer and antispermatogenic drug that exerts a large number of effects on tumor cells and germ cells. Sexually mature male Sprague-Dawley rats were housed at 22 degrees C on a 12-h light/12-h dark cycle 1 week before the experiments, with free access to food and water. LND was suspended in 0.5% methylcellulose at a concentration of 10 mg/mL and administered orally at the dose of 10 mL/kg (b.w.) as a single dose. Control rats received an equal amount of vehicle. Testes were removed, fixed for 24 h in 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium phosphate (pH 7.2 at 22 degrees C), rinsed with the same buffer, and stored at room temperature. From each sample, a block of tissue was removed by sectioning through the organ. After dehydration in ethanol at increasing concentrations (70-100%), each block was embedded in paraffin and serial 5 mm thick sections were cut using a rotatory microtome. The immunoreactivity for NTs has been observed in spermatogonia of untreated rats, while the rats treated with LND showed an immunohistochemical localization in all the stages of germinal cells. The generally well-expressed immunoreactivity for the neurotrophins receptors in treated rats observed in our study is presumably attributable to alterations of the receptors' structure and/or expression leading to changes of the activity, affinity, localization or protein interactions that may depend on sensitization of ion channels (induced by LND). Neurotrophins (NTs) appear to be interesting proteins for the modulation of sperm maturation and motility with a prominent role for the nerve growth factor (NGF), that may exert an autocrine or paracrine role. We therefore investigated the location and distribution of immunoreactivity for some neurotransmitters (SP, VIP, CGRP, nNOS, Chat), neurotrophins (NGF, BDNF, NT-3) and their own receptors (TrKA, TrKB, TrKC, p75) in the seminiferous tubules of male rats treated by LND in the light of the literature on this topic.

Ultrastructural characterization of calcification onset and progression in subdermally implanted aortic valves. Histochemical and spectrometric data.

Detailed characterization of the subdermal model is a significant tool for better understanding of calcification mechanisms occurring in heart valves. In previous ultrastructural investigation on six-week-implantated aortic valve leaflets, modified pre-embedding glutaraldehyde-cuprolinic-blue reactions (GA-CB) enabled sample decalcification with concurrent retention/staining of lipid-containing polyanionic material, which lined cells and cell-derived matrix-vesicle-like bodies (phthalocyanin-positive layers: PPLs) co-localizing with the earliest apatite nucleation sites. Additional post-embedding silver staining (GA-CB-S) revealed PPLs to contain calcium-binding sites. This investigation concerns valve leaflets subjected to shorter implantation times to shed light on the modifications associated with PPLs generation and calcification onset/progression. Spectrometric estimations revealed time-dependent calcium increase, for unreacted samples, and copper modifications indicating an increase in acidic, non-glycanic material, for GA-CB-reacted samples. Two-day-implant thin sections showed emission and subsequent reabsorption of lamellipodium-like protrusions by cells, originating ECM-containing vacuoles, and/or degeneration stages characterized by the appearance of GA-CB-S-reactive, organule-derived dense bodies and progressive dissolution of all cell membranes. In one-week-implants, the first PPL-lined cells were found to co-exist with cells where GA-CB-S-reactive material accumulated, or exudated towards their edges, or outcropped at the ECM milieu, so acquiring PPL features. PPL-derived material was observed increasingly to affect the ECM on thin sections of one-week- to six-week-implants. These results show an endogenous source for PPLs and reveal that a peculiar cascade of cell degenerative steps is associated with valve mineralization in the subdermal model, providing new useful parameters for more reliable comparison of this experimental calcification process versus the physiological and pathological processes.

Incommmensurability and unconventional superconductor to insulator transition in the hubbard model with bond-charge interaction.

We determine the quantum phase diagram of the one-dimensional Hubbard model with bond-charge interaction X in addition to the usual Coulomb repulsion U>0 at half-filling. For large enough X

Isolation of intact aortic valve scaffolds for heart-valve bioprostheses: extracellular matrix structure, prevention from calcification, and cell repopulation features.

Extracellular matrix (ECM) scaffolds isolated from valvulated conduits can be useful in developing durable bioprostheses by tissue engineering provided that anatomical shape, architecture, and mechanical properties are preserved. As evidenced by SEM, intact scaffolds were derived from porcine aortic valves by the combined use of Triton X-100 and cholate (TRI-COL) or N-cetylpyridinium (CPC) and subsequent nucleic acid removal by nuclease. Both treatments were effective in removing most cells and all the cytomembranes, with preservation of (1) endothelium basal membranes, (2) ECM texture, including the D-periodical interaction of small proteoglycans with normally D-banded collagen fibrils, and (3) mechanical properties of the treated valves. Ultrastructural features agreed with DNA, hexosamine, and uronic acid biochemical estimations. Calcification potential, assessed by a 6-week rat subdermal model, was significantly reduced by TRI-COL/nuclease treatment. This was not true for CPC only, despite better proteoglycan preservation, suggesting that nucleic acids also are involved in calcification onset. Human fibroblasts, used to repopulate TRI-COL samples, formed mono- or multilayers on surfaces, and groups of cells also were scattered within the valve leaflet framework. A biocompatible scaffolds of this kind holds promise for production of durable valve bioprostheses that will be able to undergo probable turnover and/or remodeling by repopulating recipient cells.

Malachite green and phthalocyanine-silver reactions reveal acidic phospholipid involvement in calcification of porcine aortic valves in rat subdermal model.

Subdermal implant models are helpful in the study of calcification "in vivo" and for testing anticalcific treatments. After implantation of porcine aortic valve leaflets in rat subcutis, we previously found that glutaraldehyde-Cuprolinic blue reactions (GA-CB) at low pH induce favourable tissue unmasking from mineral deposits, and visualize peculiar, electrondense layers that outline the calcifying cells and matrix vesicle-like structures. The layer-forming material seemed to consist of acidic phospholipids because of its anionic nature and differential susceptibility to chemical/enzymatic extractivity. In the present investigation, pre-embedding glutaraldehyde-Malachite green (GA-MG) reactions and subsequent osmium post-fixation were compared with pre-embedding GA-CB reactions, combined with post-embedding von Kossa silver staining (GA-CB-S), to assess whether the layer-forming material is actually composed of acidic phospholipids and exhibits calcium-binding properties. After lowering standard pH, GA-MG reactions also caused sample demineralization and the appearance of pericellular osmium-MG-reactive layers comparable to CB-reactive ones. Moreover, GA-CB-S reactions showed that major silver precipitation was superimposed to the CB-reactive layers, whereas minor metal extra-precipitation occurred at three distinct, additional sites. These results demonstrate that a unique process of cell degeneration occurs in this calcification model, in which acidic phospholipids accumulate at cell surface, replacing cell membrane and acting as major apatite nucleator. However, the overall observations are consistent with the hypothesis that certain phases are common to the various types of normal and/or abnormal calcification.

Ab-interno trabeculo-canalectomy: surgical approach and histological examination.

PURPOSE: To evaluate, on eye bank eyes, a new surgical approach aimed at removing a quadrant of the trabecular meshwork (TM), with an ab interno approach. METHODS: Gonioscopically controlled ab interno removal of the TM was done with a subretinal forcep on six human bank eyes. Serial histological sections were obtained from the treated and untreated part of each globe to assess the effect of the technique on intraocular tissues. RESULTS: Under the gonioscope, the TM was easily removed in strings of varying length. Histological examination showed, unexpectedly, that this resulted in a well-defined deep furrow in the middle of the trabecular region involving both the TM and the inner wall of Schlemm's canal. The operation created a direct communication between the anterior chamber and Schlemm's canal lumen without any evident damage to the outer canal wall and adjacent ocular structures such as the iris base and corneal endothelium. CONCLUSIONS: Our small series on human bank eyes showed that the procedure involves both the TM and the inner wall of Schlemm's canal and is therefore called ab interno trabeculocanalectomy (AITC). The intraoperative findings and the histological evidence are encouraging, and suggest that the procecedure could have potential clinical application.

Copper retention, calcium release and ultrastructural evidence indicate specific Cuprolinic Blue uptake and peculiar modifications in mineralizing aortic valves.

Previously, reactions with copper phthalocyanines at 0.05 M critical electrolyte concentration were found to cause demineralization in calcifying porcine aortic valves after subdermal implantation in rat, as well as simultaneous visualization of peculiar phthalocyanine-positive layers around cells and cell-derived matrix vesicles. In the present investigation, an appraisal was made of the mechanism and specificity of reactions with Cuprolinic Blue by comparing quantitatively calcium release and copper retention by calcified aortic valves reacted with this phthalocyanine under different critical electrolyte concentration conditions, and the corresponding ultrastructural patterns. It was found that (i) decalcifying properties are inversely proportional to salt molarity; (ii) reactivity to Cuprolinic Blue is critical electrolyte concentration-dependent, since the greatest copper retention occurred in 0.05 M critical electrolyte concentration Cuprolinic Blue-reacted samples, the only ones that also exhibited phthalocyanine-positive layers; (iii) the appearance of phthalocyanine-positive layers depends on Cuprolinic Blue uptake, revealing pericellular clustering of calcium-binding, anionic molecules; and (iv) minor Cuprolinic Blue uptake occurs by residual proteoglycans which still remain in the extracellular matrix after 6-week-long subdermal implantation. The present results indicate that this method is appropriate for the study of mineralized tissues and illustrate peculiar tissue modifications occurring at least in the experimental conditions used here.

[Post-traumatic anosmia: description of a clinical case, proposal of a standardized protocol and medico-legal comments]. / Anosmia post-traumatica: descrizione di un caso clinico, proposta di un iter valutativo standardizzato e considerazioni medico-legali.

Man's olfactory perception is considerably limited compared to that of other animals; this sense is, however, extremely important in our social lives: it helps us to "savour" our food, it enables us to appreciate perfumes and, even more important, to pick up smells that signal a danger, such as a gas leak or a fire. We describe the clinical case of a patient with anosmia and hypogeusia that appeared immediately after suffering a concussive head injury. We go through the diagnostic protocol for medico-legal assessment of hypoanosmias previously described in the literature, which includes a clinical and an imaging section. In 9% of all anosmic patients, a traumatic event precedes the onset of the disorder, with repercussions on the olfactory channels and centres of the peripherical and/or central nervous system. The overall rate of anosmia following head injury is estimated to be around 7.5%. Among the principal causes of anosmia, those of medico-legal interest constitute 35% of the total. On the basis of our personal experience and of clinical studies conducted by other Authors, we propose that a bioptic investigation of the olfactory mucosa be added to the existing protocol. The olfactory neuroepithelium of patients suffering from post-traumatic anosmia, in fact, evidences some characteristic degenerative aspects. In conclusion, we report several comments regarding the quantification of the reduction of the olfactory function in different areas of medico-legal interest.

Novel ultrastructural features as revealed by phthalocyanine reactions indicate cell priming for calcification in subdermally implanted aortic valves.

The roles played by various determinants in physiological, pathological or experimental calcification are still unclear. In this investigation, new insights were gained into structural changes occurring in porcine aortic valves undergoing mineralization in the rat subdermal model and then subjected to reactions with cationic phthalocyanines (PHTs), at salt-critical electrolyte concentrations (CEC). PHT reactions showed decalcifying effects, depending on both acidic pH in the media employed and mineral substitution by Cuprolinic Blue (CB) itself, as well as specific reactivity which enabled the ultrastructural detection of unusual, PHT-positive layers (PPLs) encircling cells and matrix vesicles, at 0.05 M CEC conditions. Other reactions at different CEC conditions, or subsequent to enzymatical or specific extractive treatments, suggest PPL appearance is due to PHT uptake by clustered anionic phospholipids, which seem to be involved in mineral precipitation. PPLs present as a novel, reliable ultrastructural parameter indicating cell propensity in priming experimental and, possibly, pathological calcification.

[Human olfactory mucosa biopsy with endoscopic technique: clinical and structural observations on neurosensory epithelium rearrangement]. / Biopsia di mucosa olfattiva con tecnica endoscopica: rilievi clinici e osservazioni strutturali sul rinnovamento tissutale.

Optical and electron microscopy have been widely used to study the structural features of olfactory epithelium in several Vertebrate species. To date, however, understanding of histopathological alterations in the human olfactory neuroepithelium has been quite limited due to the difficulty in obtaining well preserved, intact fragments of mucosa. The recent introduction of endoscopic biopsy techniques has made it possible to analyze this epithelium in greater detail. In the present work, endoscopic biopsy has been performed on samples from 10 rhinologically healthy subjects. The technique used proved quite simple and did not present any risks or complications. Moreover, all samples were well preserved, as confirmed by histology. In addition, the histological pictures suggest that normal rearrangement of neuroepithelium is not an uniform process but takes place following a zone pattern with distinct dynamics between neurosensorial and support cells. Greater diffusion of this technique would not only make it possible to use different techniques to gain more detailed knowledge of tissue structure, ultrastructure and dynamics in human neuroepithelium, but it would also provide improved diagnostic and forensic evaluation in cases of anosmia, disosmia and hyposmia.

A model for type II collagen fibrils: distinctive D-band patterns in native and reconstituted fibrils compared with sequence data for helix and telopeptide domains.

The periodical D-band pattern is generally considered a unique ultrastructural feature shared by all fibril-forming collagens, which correlates with the intrafibril, paracrystalline array of tropocollagen monomers. Distinct band patterns have been reported, however, for collagen stained long-spacing (SLS) crystallites of genetic types I, II, and III. Moreover, D-band patterns of negatively stained, native type II collagen fibrils were found to be not identical to those of type I in our previous research. Because of (a) these distinctive features, (b) tropocollagen heterotrimeric conditions (type I) vs homotrimeric conditions (type II), and (c) different lengths and poor homology between extrahelical telopeptides, the molecular array or telopeptide conformation within the extensively studied type I collagen fibrils could be not the same as those in the very much less intensively studied type II collagen fibrils. In this investigation, a distinctive positive-staining D-band pattern was found for type II collagen fibrils obtained from human cartilages. A fibril model was developed by analyzing actual D-band patterns, and matching them against simulated patterns based on the primary structure of extrahelical and helical domains in human type II tropocollagen. In particular, a more prominent b(1) band was apparent in native type II collagen fibrils than in type I. This distinctive feature was also observed for native-type collagen fibrils reconstituted from purified type II collagen, i.e., free from associated minor type XI collagen. On modeling possible monomer arrays, the best fit between microdensitograms and simulation traces was found for 234 amino acid staggering, as is also the case for type I collagen fibrils. On comparing this model with an analogous one for type I collagen fibrils, there was a higher intraband distribution of charged residues for band b(1), consistent with the higher electrondensity observed for this band in type II collagen fibrils. N- and C-telopeptide displacement in the model corresponded to D-locations of a c(2) subband, which we named c(2.0), and band a(3), respectively. In simulation profiles, c(2.0) -like and a(3) -like peaks mimicked the corresponding peaks in microdensitograms when molecular reversals were adopted at positions 10N-12N, 12C-14C, and 17C-19C for N- and C-telopeptides. Hydrophobic interactions and algorithmic predictions of protein secondary structure, according to Chou and Fasman and Rost and Sander criteria, were consistent with these conformational models, and suggest that an additional molecular reversal may occur at positions 3N-5N. These telopeptide "S-fold" conformations, interpreted as axial projections of tridimensional conformation, may represent starting points for further investigation into the still unresolved tridimensional conformation of telopeptides in monomers arrayed within type II collagen fibrils.

Cellular receptors for sex steroids in human pituitary adenomas.

Cellular receptors for sex steroids (SSRs) were studied in an unselected series of 55 human pituitary tumors. Cytosolic receptors for estrogen (ERcs) and progesterone (PgRcs) were determined in all cases and cytosolic androgen receptors (ARcs) in 47 cases. Nuclear receptors (ERns, PgRns, ARns) were also studied in 33 cases. ERs and PgRs were determined by an ELISA and ARs by [3H]methyltrienolone binding. Where both cytosolic and nuclear receptors were studied (n = 33), ERs, PgRs and ARs were found in at least one subcellular fraction in 66.7, 60.6 and 81.8% of cases respectively, ERs and ARs being mainly recovered from the cytosol and PgRs from the nucleus. No linear correlation was found between pre-operative plasma steroid hormones and their specific cellular receptors. Nonetheless, the differential expression of SSRs according to sex and gonadal status at the time of surgery strongly supports their regulation by the steroid environment in vivo: PgRcs were more frequent in tumors found in women (41.4 vs 15.4%, P < 0.05), whereas a high expression of ERcs and ARcs (> 15 fmol/mg protein) was more common in tumors found in men (34.5 vs 10.3%, P < 0.05 and 54.5 vs 24.0% respectively). PgRs were positively correlated with ERns, indicating the possibility of estrogen priming of their expression, and negatively correlated with ARs in nuclear fractions. SSRs appeared to be widely distributed among pituitary tumors, although, compared with other hormone-secreting groups, prolactinomas displayed a higher ERc expression (34.8 +/- 11.3 vs 4.8 +/- 5.1 fmol/mg protein, P = 0.007) and gonadotroph cell adenomas lower ARc values (1.3 +/- 0.8 vs 38.2 +/- 10.6 fmol/mg protein, P = 0.048). Microadenomas were characterized by a higher PgR expression than macroadenomas, whereas hemorrhagic (macro)adenomas were characterized by a high ER expression (> 90%). The present results indicate that most pituitary tumors are targets for sex steroids, SSR expression being partially triggered by the steroid environment itself. Possible physiopathological and therapeutic implications of these findings are discussed.

Liver metastases from gastric carcinoma: report of a patient treated with adoptive immunotherapy (tumor-infiltrating lymphocytes plus interleukin-2 and subsequently local-regional lymphokine-activated killer cells plus interleukin-2).

A 37-year-old patient with liver metastases from gastric cancer was treated with a double adoptive immunotherapy regimen comprising tumor-infiltrating lymphocytes plus interleukin-2 and subsequently local-regional lymphokine-activated killer cells plus interleukin-2 because of an extremely high in vitro cytotoxic specific activity on established gastric cancer cell lines. The necrosis verified in the center of the hepatic metastasis would appear to demonstrate treatment efficacy, but no clinical response was seen. In vitro cytotoxicity data alone are insufficient to predict the clinical efficacy of adoptive immunotherapy.

Cartilage type II collagen fibrils show distinctive negative-staining band patterns differences between type II and type I unfixed or glutaraldehyde-fixed collagen fibrils.

The cross striation of native and reconstituted collagen fibrils is believed to conform to a unique D-band pattern independently of the genetically distinct types of fibril-forming collagens. This investigation focuses on type II native collagen fibrils, whose negative-staining patterns are shown to differ from the usual banding exhibited by type I collagen fibrils. Negative staining with phosphotungstic acid, pH 7.4, was carried out on a) unfixed and b) glutaraldehyde-fixed collagen fibrils isolated from bovine hyaline cartilages. The band patterns obtained and their microdensitograms were compared to similarly processed type I collagen fibrils isolated from bovine fibrous tissues. Only minor differences were observed in unfixed fibrils. In the intraperiod light zones of type II fibrils, two dark bands (interbands X2-Y4 and Y4-Y2) showed different intensities with respect to their homologous bands in type I fibrils. In contrast, a marked difference was shown by glutaraldehyde-fixed fibrils. In comparison with type I fibrils, the greater stain exclusion capacity of type II fibrils yielded both the appearance of supernumerary bands, which altered banding in two intraperiod regions, and differences in the intensity of several bands in three intraperiod regions where the band distribution was similar. This stain exclusion pattern may be accounted for by molecular extradensity. The possibility that it depends on linkage with a higher number of glutaraldehyde residues and/or the persistence of cross-linked collagenic or non-collagenic proteins is discussed. To refer to the glutaraldehyde-induced band patterns in negatively stained type II and type I collagen fibrils, the terms "bands GA(II) 1-12" and "bands GA(I) 1-15," respectively, are proposed.