Marina Protasoni

The collagenic architecture of human dura mater

OBJECT Human dura mater is the most external meningeal sheet surrounding the CNS. It provides an efficient protection to intracranial structures and represents the most important site for CSF turnover. Its intrinsic architecture is made up of fibrous tissue including collagenic and elastic fibers that guarantee the maintenance of its biophysical features. The recent technical advances in the repair of dural defects have allowed for the creation of many synthetic and biological grafts. However, no detailed studies on the 3D microscopic disposition of collagenic fibers in dura mater are available. The authors report on the collagenic 3D architecture of normal dura mater highlighting the orientation, disposition in 3 dimensions, and shape of the collagen fibers with respect to the observed layer. METHODS Thirty-two dura mater specimens were collected during cranial decompressive surgical procedures, fixed in 2.5% Karnovsky solution, and digested in 1 N NaOH solution. After a routine procedure, the specimens were observed using a scanning electron microscope. RESULTS The authors distinguished the following 5 layers in the fibrous dura mater of varying thicknesses, orientation, and structures: bone surface, external median, vascular, internal median, and arachnoid layers. CONCLUSIONS The description of the ultrastructural 3D organization of the different layers of dura mater will give us more information for the creation of synthetic grafts that are as similar as possible to normal dura mater. This description will be also related to the study of the neoplastic invasion.

Early-stage microvascular alterations of a new model of controlled cortical traumatic brain injury: 3D morphological analysis using scanning electron microscopy and corrosion casting

OBJECT This study was performed to study the microvascular changes that occur during the first 12 hours after traumatic brain injury (TBI) using the corrosion casting technique. METHODS The authors performed a qualitative and quantitative morphological study of the changes in cerebral vessels at acute (3 hours) and subacute (12 hours) stages after experimental TBI. They used a model of controlled cortical impact (CCI) injury induced by a recently developed electromagnetic device (impactor), focusing their observations mainly on the microvascular alterations responsible for the formation and maintenance of tissue edema and consequent brain swelling during the first hours after TBI. They used corrosion casting, scanning electron microscopy (SEM), light microscopy, and transmission electron microscopy (TEM) to obtain a morphological qualitative map with both 2D and 3D details. RESULTS Scanning electron microscopy analysis of vascular casts documented in 3 dimensions the typical injuries occurring after a TBI: subdural, subarachnoid, and intraparenchymal hemorrhages, along with alterations of the morphological characteristics and architecture of both medium-sized and capillary vessels, including ectasia of pial vessels, sphincter constrictions at the origin of the perforating vessels, focal swelling of perforating vessels, widening of intercellular junctions, and some indirect evidence of structural impairment of endothelial cells. All of these vascular alterations were confirmed in 2D analyses using light microscopy and TEM. CONCLUSIONS The corrosion casting-SEM technique applied to a CCI experimental model proved to be a reliable method for studying the pathophysiology of the vascular alterations occurring at acute and subacute stages after CCI injury. It was also possible to obtain topographical localization of the vascular and cellular events that usually lead to hyperemia, edema, and brain swelling. Moreover, by applying informatic software to anatomical images it was possible to perform quantification and statistical analysis of the observed events.

Impact of mechanical ventilation and fluid load on pulmonary glycosaminoglycans.

The combined effect of mechanical ventilation and fluid load on pulmonary glycasaminoglycans (GAGs) was studied in anaesthetized rats ((BW 290±21.8 (SE)g) mechanically ventilated for 4h: (a) at low (∼7.5mlkg(-1)) or high (∼23mlkg(-1)) tidal volume (V(T)) and zero alveolar pressure; (b) at low or high V(T) at 5cmH(2)O positive end-expiratory pressure (PEEP); (c) with or without 7mlkg(-1)h(-1) intravenous infusion of Phosphate Buffer Solution (PBS). Compared to spontaneous breathing, GAGs extractability decreased by 52.1±1.5% and 42.2±7.3% in not-infused lungs mechanically ventilated at low V(T) or at high V(T) and PEEP, respectively. In contrast, in infused lungs, GAGs extractability increased by 56.1±4.0% in spontaneous ventilation and PEEP and up to 81.1% in all mechanically ventilated lungs, except at low V(T) without PEEP. In the absence of an inflammatory process, these results suggest that PEEP was protective at low but not at high V(T) when alveolar structures experience exceedingly high stresses. When combined to mechanical ventilation, fluid load might exacerbate edema development and lung injury.

Evidence of a discrete axial structure in unimodal collagen fibrils.

The collagen fibrils of cornea, blood vessel walls, skin, gut, interstitial tissues, the sheath of tendons and nerves, and other connective tissues are known to be made of helically wound subfibrils winding at a constant angle to the fibril axis. A critical aspect of this model is that it requires the axial microfibrils to warp in an implausible way. This architecture lends itself quite naturally to an epitaxial layout where collagen microfibrils envelop a central core of a different nature. Here we demonstrate an axial domain in collagen fibrils from rabbit nerve sheath and tendon sheath by means of transmission electron microscopy after a histochemical reaction designed to evidence all polysaccharides and by tapping-mode atomic force microscopy. This axial domain was consistently found in fibrils with helical microfibrils but was not observed in tendon, whose microfibrils run longitudinal and parallel.

Human adipose-derived stem cells promote vascularization of collagen-based scaffolds transplanted into nude mice

Aim: After in vivo implantation of cell-loaded devices, only the cells close to the capillaries can obtain nutrients to maintain their functions. It is known that factors secreted by stem cells, rather than stem cells themselves, are fundamental to guarantee new vascularization in the area of implant. Materials & methods: To investigate this possibility, we have grafted mice with Bilayer and Flowable Integra® scaffolds, loaded or not with human adipose-derived stem cells. Results: Our results support the therapeutic potential of human adipose-derived stem cells to induce new vascular networks of engineered organs and tissues. Conclusion: This finding suggests that our approach can help to form new vascular networks that allow sufficient vascularization of engineered organs and tissues in cases of difficult wound healing due to ischemic conditions.

Effects of an entomopathogen nematode on the immune response of the insect pest red palm weevil: Focus on the host antimicrobial response.

Relationships between parasites and hosts can be drastic, depending on the balance between parasite strategies and the efficiency of the host immune response. In the case of entomopathogenic nematodes and their insect hosts, we must also consider the role of bacterial symbionts, as the interaction among them is tripartite and each component plays a critical role in death or survival. We analyzed the effects induced by the nematode-bacteria complex Steinernema carpocapsae, against red palm weevil (RPW) larvae, Rhynchophorus ferrugineus. We examined the antimicrobial response of the insect when in the presence of nematocomplexes or of its symbionts, Xenorhabdus nematophila. In detail, we investigated the potential interference of live and dead S. carpocapsae, their isolated cuticles, live or dead bacterial symbionts and their lipopolysaccharides, on the synthesis and activity of host antimicrobial peptides. Our data indicate that both live nematodes and live bacterial symbionts are able to depress the host antimicrobial response. When nematodes or symbionts were killed, they lacked inhibitory properties, as detected by the presence of antimicrobial peptides (AMPs) in the host hemolymph and by assays of antimicrobial activity. Moreover, we isolated S. carpocapsae cuticles; when cuticles were injected into hosts they revealed evasive properties because they were not immunogenic and were not recognized by the host immune system. We observed that weevil AMPs did not damage X. nematophila, and the lipopolysaccharides purified from symbionts seemed to be non-immunogenic. We believe that our data provide more information on the biology of entomopathogenic nematodes, in particular concerning their role and the activity mediated by symbionts in the relationship with insect hosts.

Role of glutamatergic neurotransmission in the enteric nervous system and brain-gut axis in health and disease

Several studies have been carried out in the last 30 years in the attempt to clarify the possible role of glutamate as a neurotransmitter/neuromodulator in the gastrointestinal tract. Such effort has provided immunohistochemical, biomolecular and functional data suggesting that the entire glutamatergic neurotransmitter machinery is present in the complex circuitries of the enteric nervous system (ENS), which participates to the local coordination of gastrointestinal functions. Glutamate is also involved in the regulation of the brain-gut axis, a bi-directional connection pathway between the central nervous system (CNS) and the gut. The neurotransmitter contributes to convey information, via afferent fibers, from the gut to the brain, and to send appropriate signals, via efferent fibers, from the brain to control gut secretion and motility. In analogy with the CNS, an increasing number of studies suggest that dysregulation of the enteric glutamatergic neurotransmitter machinery may lead to gastrointestinal dysfunctions. On the whole, this research field has opened the possibility to find new potential targets for development of drugs for the treatment of gastrointestinal diseases. The present review analyzes the more recent literature on enteric glutamatergic neurotransmission both in physiological and pathological conditions, such as gastroesophageal reflux, gastric acid hypersecretory diseases, inflammatory bowel disease, irritable bowel syndrome and intestinal ischemia/reperfusion injury.

The Cutaneous Microvascular Architecture of Human Diabetic Toe Studied by Corrosion Casting and Scanning Electron Microscopy Analysis

In this morphological study, we report on the three‐dimensional microvascular architecture constituting the toes of a patient affected by diabetic microangiopathy. We applied corrosion casting (CC) technique to the toes of a patient affected by Type 2 diabetes, who underwent surgery for explantation of inferior left limb due to necrotic processes of soft tissues. The toes of a foot traumatically explanted in a motorcycle accident were kept as controls. According to technical protocols, toes were injected with a low‐viscosity acrylic resin (Mercox) through the major digital artery, tissues were corroded in KOH solution (8%), and resulting casts processed for SEM observations. Already at low magnification, in diabetic toes, we found an impairment of the linear track‐like disposition of the vessels of plantar side, with signs of vascular disruption and obliterations, stopped resin, and leakages. Capillaries under the nail and a lot of vascular villi in eponychium and nail borders were damaged, and vascular regression phenomena acting on them were clearly visible. Resin leakages and impairment of normal vascular architecture were also observed in the root of the nail. This preliminary report represents only the first step for further investigations regarding morphological three‐dimensional appearance of diabetic microangiopathy. CC and scanning electron microscopy technique well documented these morphological modifications, highlighting on both structural and ultrastructural features of diabetic toes microvessels. In conclusion, our qualitative data try to better focus on the pathophysiological mechanisms involved in diabetic dermopathy and microangiopathy, proposing CC as useful method to investigate on them. Anat Rec 293:1639–1645, 2010.

Atherosclerotic alterations in human carotid observed by scanning electron microscopy

Atherosclerosis involves all the layers of the artery wall, but the events involving the intimal portion are fundamental to understand the evolution and gravity of lesions. This study shows that scanning microscopy is instrumental for better understanding the physiopathology of this disease.

Not only tendons: The other architecture of collagen fibrils

For many decades the fibrillar collagens have been the subject of a remarkable body of ultrastructural research. The vast majority of the studies, however, were carried out on tendon or on tendon-derived material. For many reasons this reflects an obvious choice but at the same time it also is an unfortunate circumstance, because this flooding of tendon-related data can easily encourage the false confidence that all connective tissues are similar. The reality is quite the opposite, and a different fibrillar structure has been long time observed on collagen fibrils from different tissues, the most notable example being offered by corneal fibrils. The same architecture can be found in a number of disparate tissues and may actually be the prevalent one on a whole-body scale. Although these fibrils diverge from those of tendon in their architecture, size, D-period, composition, cross-linking and fibrillogenesis mechanism, their structure was the subject of rather sparse ultrastructural studies and even today their mere existence is often overlooked or ignored. This paper summarizes the main aspects of the structural biology of these forgotten fibrils.