Towards a common language in neurosurgical outcome evaluation: the NEON (NEurosurgical Outcome Network) proposal.

BACKGROUND: The aim of this study was to achieve a consensus on the minimum set of outcome measures and predictors to be used in the neurosurgical practice and on the timing of outcome assessment. METHODS: A consensus building approach was employed. All neurosurgical departments in Lombardy (Italy) were invited to participate by the Carlo Besta Neurologic Institute IRCCS Foundation. Three workshops were organized during which a multidisciplinary group called Neurosurgical Outcome Network (NEON) was created and the methodology to select outcome measures, predictors, and timing of outcome assessment was established. Eight working groups were created for the different neurosurgical diseases (neuro-oncological, skull base, vascular, traumatic, spinal, peripheral nervous system, malformation, functional) and 8 workshops were organized to identify the outcome measures and predictors specific for each of the neurosurgical diseases based on the experts' clinical practice and the existing literature. RESULTS: A total of 20 neurosurgical departments participated in this study. Specific outcome measures, predictors and the timing of outcome assessment were identified for each of the 8 neurosurgical diseases. Moreover, a list of variables common to all pathologies were identified by the NEON group as further data to be collected. CONCLUSIONS: A consensus on the minimum set of outcome measures and predictors and the timing of outcome assessments for 8 neurosurgical diseases was achieved by a group of neurosurgeons of the Lombardy region, called NEON. These sets could be used in future studies for a more homogeneous data collection and as a starting point to reach further agreement also at national and international level.

Blockade of IGF2R improves muscle regeneration and ameliorates Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a debilitating fatal X-linked muscle disorder. Recent findings indicate that IGFs play a central role in skeletal muscle regeneration and development. Among IGFs, insulinlike growth factor 2 (IGF2) is a key regulator of cell growth, survival, migration and differentiation. The type 2 IGF receptor (IGF2R) modulates circulating and tissue levels of IGF2 by targeting it to lysosomes for degradation. We found that IGF2R and the store-operated Ca2+ channel CD20 share a common hydrophobic binding motif that stabilizes their association. Silencing CD20 decreased myoblast differentiation, whereas blockade of IGF2R increased proliferation and differentiation in myoblasts via the calmodulin/calcineurin/NFAT pathway. Remarkably, anti-IGF2R induced CD20 phosphorylation, leading to the activation of sarcoplasmic/endoplasmic reticulum Ca2+ -ATPase (SERCA) and removal of intracellular Ca2+ . Interestingly, we found that IGF2R expression was increased in dystrophic skeletal muscle of human DMD patients and mdx mice. Blockade of IGF2R by neutralizing antibodies stimulated muscle regeneration, induced force recovery and normalized capillary architecture in dystrophic mdx mice representing an encouraging starting point for the development of new biological therapies for DMD.

Arterial and microvascular supply of cerebral hemispheres in the nude mouse revealed using corrosion casting and scanning electron microscopy.

Morphological analyses of cerebral vascularization are not only important for the characterization of the anatomical and physiological relationships between vascular and nervous tissue, but also required to understand structural modifications that occur in many pathological conditions affecting the brain. The aim of this study was to generate a three-dimensional vascular map of the cerebral hemispheres in the nude mouse brain, a widely used animal model for studying tumour biology. We used the corrosion casting (CC) technique to isolate blood vessels from 30 nude mouse brains. All casts were analysed using scanning electron microscopy (SEM), which generated quantitative data regarding vessel length and diameter as well as inter-vascular and inter-branching distances. We identified three different topographical regions: (i) the cortical region, characterized by a superficial wide sheet of vessels giving rise to terminal perforant vessels that penetrate the grey matter; (ii) the inner part of the grey matter, in which dense capillary nets form many flake-like structures extending towards the grey-white matter boundary, where perforant vessels finally change direction and form a well-defined vascular sheet; and (iii) the white matter layer, characterized by a more disorganized vascular architecture. In this study, we demonstrate the accuracy of the CC-SEM method in revealing the 3D-topographical organization of the vascular network of the normal nude mouse brain. These baseline data will serve as a reference for future anatomical investigations of pathological alterations, such as tumour infiltrations, using the nude mouse model.

Infrequent Hemorrhagic Complications Following Surgical Drainage of Chronic Subdural Hematomas.

Chronic subdural hematomas mainly occur amongst elderly people and usually develop after minor head injuries. In younger patients, subdural collections may be related to hypertension, coagulopathies, vascular abnormalities, and substance abuse. Different techniques can be used for the surgical treatment of symptomatic chronic subdural hematomas : single or double burr-hole evacuation, with or without subdural drainage, twist-drill craniostomies and classical craniotomies. Failure of the brain to re-expand, pneumocephalus, incomplete evacuation, and recurrence of the fluid collection are common complications following these procedures. Acute subdural hematomas may also occur. Rarely reported hemorrhagic complications include subarachnoid, intracerebral, intraventricular, and remote cerebellar hemorrhages. The causes of such uncommon complications are difficult to explain and remain poorly understood. Overdrainage and intracranial hypotension, rapid brain decompression and shift of the intracranial contents, cerebrospinal fluid loss, vascular dysregulation and impairment of venous outflow are the main mechanisms discussed in the literature. In this article we report three cases of different post-operative intracranial bleeding and review the related literature.

Isolation and expansion of human and mouse brain microvascular endothelial cells.

Brain microvascular endothelial cells (BMVECs) have an important role in the constitution of the blood-brain barrier (BBB). The BBB is involved in the disease processes of a number of neurological disorders in which its permeability increases. Isolation of BMVECs could elucidate the mechanism involved in these processes. This protocol describes how to isolate and expand human and mouse BMVECs. The procedure covers brain-tissue dissociation, digestion and cell selection. Cells are selected on the basis of time-responsive differential adhesiveness to a collagen type I-precoated surface. The protocol also describes immunophenotypic characterization, cord formation and functional assays to confirm that these cells in endothelial proliferation medium (EndoPM) have an endothelial origin. The entire technique requires ∼7 h of active time. Endothelial cell clusters are readily visible after 48 h, and expansion of BMVECs occurs over the course of ∼60 d.

Human and mouse brain-derived endothelial cells require high levels of growth factors medium for their isolation, in vitro maintenance and survival.

BACKGROUND: Brain microvascular endothelial cells (BMVECs) constitute the primary limitation for passage of ions and molecules from the blood into the brain through the blood brain barrier. Numerous multi-step procedures for isolating and culturing BMVECs have been described. However, each one demonstrates major limitations in purity of culture and/or low proliferation rate. Our goal was to study the efficiency of our pending patent medium, Endothelial Proliferation Medium (EndoPM), on the isolation and purification of human and murine BMVECs. METHODS: BMVECs, cultured in EndoPM were compared to those cultured in a commercial medium EBM. Cultures were characterized by flow cytometric analysis, lineage differentiation, the ability to form tube-like structure, immunofluorescence, molecular analyses and also in an in vivo model assay. Moreover permeability was assayed by monitoring the passage of Dextran-FITC through a tight monolayer of BMVECs grown to confluence in Boyden chambers. One way Anova two-tailed test was utilized for all statistical analyses. RESULTS: The properties of ECs in human and murine BMVECs is confirmed by the expression of endothelial markers (CD31, CD105, CD146, Tie-2 and vWF), of representative proangiogenic genes (ICAM1, VCAM1 and integrin ITGAV), of considerable tube-forming ability, with low-density lipoprotein uptake, eNOS and GLUT-1 expression. Furthermore cells are able to express markers of the junctional architecture as VE-cadherin, ß-catenin and Claudin-5 and greatly reduce dextran permeability as barrier functional test. Moreover BMVECs spontaneously organize in vascular-like structures and maintain the expression of endothelial markers in an in vivo xenograft model assay. The significant effect of EndoPM is confirmed by the study of proliferation index, survival index and the behaviour of BMVECs and fibroblasts in co-culture conditions. CONCLUSION: Herein we describe a simple and reproducible method for the isolation and expansion of human and mouse BMVECs, based on a newly formulated medium (EndoPM) with optimized concentration of growth factors (EGF, FGF-2 and Bovine Brain Extract-BBE). This procedure should facilitate the isolation and expansion of human and mouse BMVECs with extended lifetime, good viability and purity. This approach may provide an effective strategy to aid phenotypical and functional studies of brain vessels under physiological and pathological conditions.

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.

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.

Pulsed radiofrequency effects on the lumbar ganglion of the rat dorsal root: a morphological light and transmission electron microscopy study at acute stage.

Since the dorsal root ganglia represent the first structure of pain modulation, they are the target of the newest therapies of neuropathic pain. Between these, pulsed radiofrequency (PRF) has been described among the promising non-invasive methods. Although the results encourage the clinical use of this procedure, their mechanism of action is still unclear. Aim of our study was to analyze acute effects of PRF on the rat lumbar ganglion and on nervous fibres running inside it. Clinical works describe PRF treatment as a technique without any visible neurological deficit. The few disposable histological works are contractictory: some describe no signs of cellular damage and some demonstrate visible intracellular modifications. A total of 20 male Wistar rats were deeply anesthesized. Ten were positioned in a stereotactic system, and exposed to PRF at 2 Hz for 30 s after exposition of paravertebral muscles and positioning of a stimulation needle on left L4 ganglion. The other ten were used as controls. After 1 h, the left dorsal root ganglions L3, L4, L5 of the 20 animals were explanted, fixed in 2.5% Karnowsky solution and prepared for light and transmission electron microscopy. At light microscopy no differences between treated and control animals were observed; at transmission electron microscopy, instead, it was possible to observe that T gangliar cells contained an abnormal abundant smooth reticulum with enlarged cisternae and numerous vacuoles; myelinated axons presented pathological features and their myelin coverage was not adherent. Instead, unmyelinated axons appeared normal in shape and dimension and the Schwann cells surrounding it had intact plasmamembrane. Our results, obtained at acute stage, reveal that the PRF procedure should destroy the myelin envelope of nervous fibres. Further future studies, at chronic stage, should give other information on the prognosis of the myelinic damage.

Intrathecal baclofen does not inhibit the growth of different bacterial species and Candida albicans.

BACKGROUND: The antimicrobial activity of intrathecal baclofen was investigated. Several different microorganisms were used: Staphylococcus aureus (beta-lactamase-positive and beta-lactamase-negative strains); S epidermidis; Enterococcus faecalis; Klebsiella pneumoniae; Escherichia coli; Pseudomonas aeruginosa; and Candida albicans. METHODS: Three experimental approaches were used to assess baclofen antimicrobial activity: (1) determination of the MIC; (2) determination of the MBC; and (3) kinetic time-kill assay. Experiments were performed according to current methods of the NCCLS. RESULTS: As compared with control organisms exposed to physiologic saline, organisms exposed to baclofen over a 10-day period failed to reduce the number of viable cells by at least 3 log(10), as requested by NCCLS criteria. CONCLUSIONS: Because the viability of the investigated organisms was not reduced over that of microbial suspensions exposed to physiologic saline, we conclude that intrathecal baclofen has no measurable activity against different bacterial species and C albicans.

Baclofen and potential therapeutic use: studies of neuronal survival.

Up to now, baclofen (a GABA(B) receptor agonist) has been used for the treatment of severe spasticity unresponsive to oral antispasmodics. Although in humans it is usually administered at 2 mg/ml, the dosage to be used in the treatment of other diseases is unknown. For this reason, it is important to determine the safe maximum dosage and toxicity at the clinically used concentration. Primary cortical neurons represent a useful model to test the safety of baclofen. We performed a colorimetric assay (MTT test) as well as electron microscopy investigations, to determine neuronal survival after the treatment with baclofen at a concentration of 2 and 4 mg/ml. Our results demonstrated that, in our experimental model, neither concentration affected neuronal survival. Considering the above results, we can conclude that at the used concentrations, this drug is safe and its clinical use should be encouraged.

A new method for the joint visualization of vascular structures and connective tissues: corrosion casting and 1 N NaOH maceration.

Corrosion casting combined with scanning electron microscopy (SEM) has been widely used to study the morphofunctional aspects of microcirculation in many organs. In this study, we present an optimization of the corrosion casting (CC) technique associating it with NaOH 1 N maceration method to obtain a clear visualization of the relationships existing between the microvascular architecture of an organ and its extracellular matrix. Briefly, experiments were performed macerating the tissue previously injected with a low viscosity acrylic resin in 1 N NaOH and then observing it at SEM. In this study, we present an application of this technique to better evaluate the extracellular components of the vascular wall in medium-sized and capillary vessels both in skin and in kidney. The results obtained yielded clear images of the three-dimensional layout of medium-sized and capillary vessels in comparison with the extracellular environment. Furthermore, detailed information was obtained on the three-dimensional layout of fibers constituting the walls of venules, arterioles, and capillaries. In addition, the tubular collagenic structures surrounding the excretory tubules of the kidney and the dermal glands of the skin were depicted and their relationships with their vascular supply described in detail.

Whiplash injury and oculomotor dysfunctions: clinical-posturographic correlations.

Oculomotor dysfunctions are hidden causes of invalidity following whiplash injury. Many patients with whiplash injury grade II present oculomotor dysfunctions related to input disturbances of cervical or vestibular afferents. We used static posturography to investigate 40 consecutive patients with whiplash injury grade II and oculomotor dysfunctions. We demonstrated a relation between length and surface of body sway: the surface value (A) was higher than the length value (L) and this led to an open graph of body sway in the statokinesigram. Oculomotor rehabilitation can resolve the impairment of vestibular function but if therapy is delayed or the patient has been wearing an orthopaedic neck collar, more therapeutic sessions are required. In conclusion, without rehabilitation of the oculomotor muscles other therapies are not sufficient to recover the impairment caused by whiplash injury.

The collagenic structure of human digital skin seen by scanning electron microscopy after Ohtani maceration technique.

We performed a morphological scanning electron microscope (SEM) study to describe the fine structure and disposition of collagenous tissue in the human toe. After therapeutic amputation of a human right leg, we applied the Othani maceration technique to the skin of three toes surgically explanted from the foot. We distinguished eight cutaneous regions and focused on some specialized collagenous structures differing in the thickness of the skin. The eight areas investigated were: the dorsal skin, the eponychium, the perionychium, the hyponychium, the region under the visible nail, the nail root, the plantar skin and finally the toe tip. Each of these areas is characterized by a distinctive collagenous surface disposition, with some peculiar features mostly related to dermal papillae. At high magnification, we observed the spatial arrangement of the collagen fibers constituting the top of the dermal papillae that represents the attachment site of the proliferative basal layer of the epidermis. We also noted an impressive density of collagen fibers throughout the thickness of the dermal layer, organized in specialized structures and constituting the skeleton of dermal thermoreceptorial corpuscles or sweat glands. A combination of SEM and Ohtani technique disclosed the three-dimensional architecture of the collagenous matrix of tarsal skin under physiologic conditions, giving a detailed description of the most reactive tissue during pathologic processes.

Structure and ultrastructure of microvessels in the kidney seen by the corrosion casting method.

Scanning electron microscopic observation of corrosion casts is the finest technique to describe spatial patterns of microvessels in many organs, giving a readily interpreted representation of their vascular architecture without interference from surrounding tissues. We focused on the renal cortex of guinea pigs to make an in-depth morphological analysis of structural and ultrastructural details left by the cells on the resin cast. In addition, we made a qualitative description of normal variants usually observed in glomerular disposition, arteriolar morphology or capillary arrangement in the space to shed more light on the relationship between vascular tissue and surrounding cells. The study also disclosed some examples of vascular adaption to physiological and pathological conditions occurring in renal microvessels such as many systems essential to flow regulation, filtration and excretory processes. At lower magnification, all major vessels can be readily distinguished: interlobar, arciform and interlobular arteries and veins, along with a web of peritubular and capsular capillaries. At higher magnification, the glomeruli become visible and the afferent and efferent arteries and the tortuosity the inner vessels can be distinguished. In some of them, the resin, due to the narrowing sizes, suddenly stopped leaving a half-casted glomerulus. This helped to reveal its internal circulation characterized by thin capillaries with a high degree of bi or trifurcation. In addition, we confirmed the close correspondence between cellular ultrastructural detail (pores, corrugations of cellular membrane, perivascular cell branches) and the impressions left on the resin visible only at high magnifications.