Riccardo Rodriguez y Baena

Adipose-Derived Stem Cell Therapy for Intervertebral Disc Regeneration: An In Vitro Reconstructed Tissue in Alginate Capsules

The degenerative pathologies of the intervertebral disc have a remarkable social impact in the industrialized countries and can provide serious disabilities in the population. The current treatment consists of conservative treatments (such as symptomatic pharmacological therapies and physiokinetic therapy) and surgical treatments (intervertebral fusion, total disc replacement, nucleus pulposus (NP) replacement, or surgical exeresis). Recent advances in cell therapy foresee the possibility of regenerating the damaged disc; the autologous disc tissue can be withdrawn, in vitro regenerated, and re-implanted. The aim of this work was to verify whether autologous adipose-derived adult stem cells can improve the quality of an in vitro reconstructed nucleus pulposus tissue. A three-dimensional (3D) co-culture of NP cells and adipose tissue non-adipocyte fraction cells (nAFs) was assessed in a previously developed alginate 3D culture system following the good manufacturing practice guidelines to ensure patient safety for clinical studies. Morphological investigation of cultured and co-cultured cells was performed using transmission electron microscopy and immunofluorescence for collagen type I, aggrecan, CD90, CD34, and vimentin. Results indicate that co-culture of NP and nAFs improves the quality of the in vitro reconstructed tissue in term of extracellular matrix production and 3D cell organization. Technological resources are available for NP cell encapsulation intended for regenerating the intervertebral disc.

Fractal dimension as a quantitator of the microvasculature of normal and adenomatous pituitary tissue.

It is well known that angiogenesis is a complex process that accompanies neoplastic growth, but pituitary tumours are less vascularized than normal pituitary glands. Several analytical methods aimed at quantifying the vascular system in two‐dimensional histological sections have been proposed, with very discordant results. In this study we investigated the non‐Euclidean geometrical complexity of the two‐dimensional microvasculature of normal pituitary glands and pituitary adenomas by quantifying the surface fractal dimension that measures its space‐filling property. We found a statistical significant difference between the mean vascular surface fractal dimension estimated in normal versus adenomatous tissues (P = 0.01), normal versus secreting adenomatous tissues (P = 0.0003), and normal versus non‐secreting adenomatous tissues (P = 0.047), whereas the difference between the secreting and non‐secreting adenomatous tissues was not statistically significant. This study provides the first demonstration that fractal dimension is an objective and valid quantitator of the two‐dimensional geometrical complexity of the pituitary gland microvascular network in physiological and pathological states. Further studies are needed to compare the vascular surface fractal dimension estimates in different subtypes of pituitary tumours and correlate them with clinical parameters in order to evaluate whether the distribution pattern of vascular growth is related to a particular state of the pituitary gland.

A role for the transcription factor HEY1 in glioblastoma

Glioblastoma multiforme (GBM), the highest‐grade glioma, is the most frequent tumour of the brain with a very poor prognosis and limited therapeutic options. Although little is known about the molecular mechanisms that underlie glioblastoma formation, a number of signal transduction routes, such as the Notch and Ras signalling pathways, seem to play an important role in the formation of GBM. In the present study, we show by in situ hybridization on primary tumour material that the transcription factor HEY1, a target of the Notch signalling pathway, is specifically up‐regulated in glioma and that expression of HEY1 in GBM correlates with tumour‐grade and survival. In addition, we show by chromatin immunoprecipitations, luciferase assays and Northern blot experiments that HEY1 is a bona fide target of the E2F family of transcription factors, connecting the Ras and Notch signalling pathways. Finally, we show that ectopic expression of HEY1 induces cell proliferation in neural stem cells, while depletion of HEY1 by RNA interference reduces proliferation of glioblastoma cells in tissue culture. Together, these data imply a role for HEY1 in the progression of GBM, and therefore we propose that HEY1 may be a therapeutic target for glioblastoma patients. Moreover, HEY1 may represent a molecular marker to distinguish GBM patients with a longer survival prognosis from those at high risk.

Metalloproteases and intracranial vascular lesions

Recent studies have suggested that metalloproteinases (MMP) might be involved in the pathogenesis of cerebral aneurysm formation and rupture and that elevated serum levels of MMP may effectively be considered as possible markers of cerebrovascular malformations. The present study was planned in order to verify if serum levels of MMPs may be the mirror of the MMP activity in the wall of intracranial aneurysms, reflecting the predisposition to aneurysm development and/or rupture. A series of 84 patients operated for intracranial cerebrovascular lesions (63 aneurysms and 21 arterovenous malformations (AVM)) and 20 controls entered the study. Among the 63 cases of intracranial aneurysms, nine were discovered before rupture, while 54 patients were included after subarachnoid hemorrhage (SAH). Using radioimmunoassay, plasma elastase levels were measured in all cases, while in 25 cases, when aneurysmectomy was possible, the activity of elastase and collagenase were measured in aneurysm samples. Mean plasma elastase level in patients bearing both an intracranial aneurysm or an intracranial AVM was significantly higher than in controls, while there was no significant difference between plasmatic level of elastase in patients with aneurysms when compared with patients bearing an intracranial AVM; there was no significant difference between mean elastase level in patients who suffered SAH and patients bearing an intracranial unruptured aneurysm. The activity of elastase and collagenase measured in the aneurysm wall were significantly higher in cases of ruptured than in unruptured aneurysms. The present results show that plasmatic level of elastase does not reflect the activity of MMP as measured in the aneurysm wall and that the patterns of MMP activities measured in the aneurysm wall differ considerably at different stages of SAH. This suggests that local rather than systemic changes in metalloproteases activity might be involved in cerebral aneurysm formation and rupture.

Activity of α1-antitrypsin and cigarette smoking in subarachnoid haemorrhage from ruptured aneurysm

An altered equilibrium of protease/protease-inhibitor factors may be involved in the pathogenesis of aneurysm rupture: alpha 1-antitrypsin (alpha 1-AT) represents the most relevant inhibitor of elastase, a proteolytic enzyme enhancing catabolic processes of collagen metabolism. Cigarette smoking has been shown to significantly reduce the inhibitory effect of alpha 1-AT on proteases. In the present study we test the hypothesis whether the activity of alpha 1-AT is altered in patients with subarachnoid haemorrhage (SAH) and if is there any relationship between alpha 1-AT activity and the high risk of aneurysm rupture in smokers. The patients were subdivided in the following groups: (a) patients with unruptured aneurysm (n = 10); (b) patients presenting with SAH admitted within 48 h after the episode (n = 20); (c) patients presenting with SAH admitted > 48 h after the episode (n = 14); (d) controls (n = 10): patients with neither cerebrovascular nor acute disease. Blood samples were obtained immediately at admission. Measurement of alpha 1-AT level was determined by immunoturbidimetric method. In order to obtain qualitative data about the anti-protease activity of alpha 1-AT (expressed as collagenase inhibitory percentage capacity (CIC) at different doses) we consider the 20 cases admitted for SAH within 48 h. The mean serum level of patients with unruptured aneurysms is significantly lower than that of patients with SAH (p < 0.01), while the mean serum level of alpha 1-AT in controls does not significantly differ from other groups. The mean serum level of alpha 1-AT in patients admitted > 48 h after SAH is significantly higher than that of patients admitted within 48 h after the haemorrhage (p < 0.02). Considering the smoking habit of patients, there is no significant difference in alpha 1-AT levels in each subgroup of patients. A multivariate analysis considering alpha 1-AT CIC, showed that alpha 1-AT CIC in patients with ruptured aneurysms is significantly reduced if compared to controls and unruptured aneurysms (F = 50.759; p < 0.001). Moreover, considering alpha 1-AT CIC and smoking habit in each group the covariance analysis showed that while in controls and unruptured aneurysms there is no difference in alpha 1-AT CIC between smokers and non smokers, in cases of SAH, cigarette smoking significantly influences the alpha 1-AT CIC. The present results suggest that the basic mechanism behind the increased risk of SAH in smokers involves a qualitative deficiency of alpha 1-AT.

Primary solitary intracranial melanoma: Case report and review of the literature

Among CNS tumors, intracranial melanomas represent a subject of interest for neurooncologists and neurosurgeons because clinical and radiological patterns of these tumors can mimic the presence of meningiomas, and in spite of their malignant behavior they can be satisfactorily treated. In the present report we describe a new case of primary intracranial melanoma that displayed some radiological features of meningioma; we review the clinical features of 80 previously well-documented cases. The importance of neuroradiological and histochemical (S-100 protein, antimelanin antibodies, proliferating cell nuclear antigen staining) methods and of flow cytometry in helping with histopathological examination is stressed. Review of the clinical histories demonstrates that surgical excision is recommended in most cases, depending on tumor location, and that if total removal is performed, long-term disease-free periods can be attained.

Arachidonic acid metabolism and pathophysiologic aspects of subarachnoid hemorrhage in rats.

We studied the ex vivo production of prostaglandin D2, prostaglandin E2, 6-ketoprostaglandin F1 alpha, and leukotriene C4 in the brain tissue of rats subjected to experimental subarachnoid hemorrhage. The ex vivo method allows the study of arachidonic acid metabolites released from brain slices at different times after subarachnoid hemorrhage induction and reflects the residual capacity for arachidonic acid metabolism after the pathologic event. The rats were sacrificed 30 minutes, 1 and 6 hours, and 2 days after subarachnoid hemorrhage was induced by the injection of 0.30 ml autologous arterial blood into the cisterna magna. Concentration of prostaglandin D2 and 6-ketoprostaglandin F1 alpha was increased significantly relative to control 2 days after induction. The concentration of prostaglandin E2 was increased significantly 6 hours after induction, while ex vivo production of leukotriene C4 was increased significantly at 1 and 6 hours and 2 days. The correlation between these results and the occurrence of vasospasm after subarachnoid hemorrhage is discussed. The results obtained from the ex vivo incubation of brain tissue slices after experimental subarachnoid hemorrhage suggest that after the hemorrhage there is a significant modification of brain eicosanoid metabolism, which could be of great importance in interpreting the pathogenesis of subarachnoid hemorrhage-related neuronal impairment.

Oxidative damage after severe head injury and its relationship to neurological outcome

OBJECTIVE We sought to establish the time course of reactive oxygen species after severe head injuries in humans and to investigate their relationship with clinical outcomes. METHODS Both the markers of oxidative damage—malonylaldehyde (MDA) and the enzymatic and nonenzymatic antioxidant defenses (i.e., superoxide dismutase [SOD] and vitamin E [VE], respectively)—were studied. To assess the time course of MDA, SOD, and VE, jugular bulb (JB) and peripheral venous blood samples were obtained from 30 patients within 8 hours of severe head trauma onset (T0) and 6 (T1), 12 (T2), 24 (T3), and 48 hours (T4) after trauma onset. Patients were divided into good and poor outcome groups according to their 6-month neurological outcome as determined on the basis of their Glasgow Outcome Scale scores and biochemical profiles. RESULTS In JB samples, MDA levels increased significantly at T1, T2, T3, and T4 as compared with T0; SOD activity increased significantly at T2 and T3 as compared with T0; and VE levels decreased significantly at T1, T2, and T3 as compared with T0. The same variables did not change significantly over time in peripheral venous blood samples. Moreover, the MDA levels and SOD activity detected in JB samples were significantly higher in the poor outcome group at T1 and T2. No significant difference in VE levels was observed between the two outcome groups. CONCLUSION Reactive oxygen species-mediated oxidative damage can play an important role in determining the prognosis of severe brain injury in humans.

Human glioblastoma tumours and neural cancer stem cells express the chemokine CX3CL1 and its receptor CX3CR1

Human gliomas represent an unmet clinical challenge as nearly two-thirds of them are highly malignant lesions with fast progression, resistance to treatment and poor prognosis. The most severe form, the glioblastoma multiforme, is characterised by a marked and diffuse infiltration through the normal brain parenchyma. Given the multiple effects of chemokines on tumour progression, aim of this study was to analyse the expression of the chemokine CX3CL1 and of its specific receptor CX3CR1 in 36 human surgical glioma samples, with different degrees of histological malignancy and in glioblastoma-derived neurospheres. Herein we show that both ligand and receptor are expressed at the mRNA and protein levels in most specimens (31/36). While receptor expression was similarly detected in low or high grade tumours, the uppermost scores of CX3CL1 were found in grades III-IV tumours: oligodendrogliomas, anaplastic astrocytomas and glioblastomas. Accordingly, the expression of CX3CL1 was inversely correlated with patient overall survival (p = 0.01). Glioblastoma-derived neurospheres, containing a mixed population of stem and progenitor cells, were positive for both CX3CR1 and for the membrane-bound chemokine, which was further up-regulated and secreted after TNF-IFNγ stimulation. Confocal microscopy of 3D neurospheres showed that the ligand was primarily expressed in the outer layer cells, with points of co-localisation with CX3CR1, indicating that this ligand-receptor pair may have important intercellular adhesive functions. The high expression of CXC3L1 in the most severe forms of gliomas suggests the involvement of this chemokine and its receptor in the malignant behaviour of these tumours.

Bioenergetics of different brain areas after experimental subarachnoid hemorrhage in rats.

We studied energy metabolism after experimental subarachnoid hemorrhage in rats. Four different cerebral areas were tested: frontal cortex, occipital cortex, hippocampus, and brainstem. Vmax of the following enzymatic activities was evaluated: in the homogenate: hexokinase, phosphofructokinase, and lactate dehydrogenase for the glycolytic pathway, and glucose-6-phosphate dehydrogenase for the hexose monophosphate shunt; in the purified nonsynaptic mitochondria: NAD+-isocitrate dehydrogenase, citrate synthase, and succinate dehydrogenase for the Krebs cycle, and cytochrome oxidase for the electron transfer chain. We also evaluated some parameters related to the respiration of nonsynaptic mitochondria (State 3, State 4, uncoupled state, respiratory control ratio, and ADP:O ratio). Subarachnoid hemorrhage did not significantly affect Vmax of the enzymatic activities related to anaerobic and aerobic metabolism; however, mitochondrial respiration was affected, particularly in the presence of NADH-producing substrates (glutamate + malate).