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Eduardo Fernandez

Publications and source records attributed to Eduardo Fernandez.

At least 19 recordsLinked to original sources
PubMed2006

A method of combined single-cell electrophysiology and electroporation.

This paper describes a method of extracellular recording and subsequent electroporation with the same electrode in single retinal ganglion cells in vitro. We demonstrate anatomical identification of neurons whose receptive fields were measured quantitatively. We discuss how this simple method should also be applicable for the delivery of a variety of intracellular agents, including gene delivery, to physiologically characterized neurons, both in vitro and in vivo.

Action Potentials↗
PubMed2006

The adult "paraplegic" rat: treatment with cell graftings.

Spinal cord injury often results in irreversible and permanent neurologic deficits below the lesion level. Nowadays, treatment is limited to drugs and/or physiotherapy aimed at compensating disability. New experimental studies focus on the transplantation of cells capable of surviving, regenerating tissue, recovering functions and/or improving symptoms. A review of such type of studies on spinal cord reconstruction published between 1991 and 2004 is presented. In the latter years, cell transplantation appeared as the most promising approach in spinal cord regeneration research. To date, this promise has not been maintained, despite the appearance of new attractive cell populations for grafting, such as neural stem cells. The demonstration that stem cells exist in the adult brain and that they can be isolated and expanded in vitro offers the possibility to test such interesting cells in the paraplegic rat. Some neurotrophic factors can facilitate axonal regeneration and neuronal survival. Therefore, the development of strategies, such as implanting neural stem cells engineered to secrete neurotrophic factors directly in the lesion site, could be important to promote regeneration in the injured spinal cord. Despite all the strategies used till now, the problem of the paraplegic rat remains. Only the solution of such problem will authorize studies in higher mammals and, finally, the clinical application in human patients. The paraplegic adult rat with a T8 spinal cord transection should be considered the standard experimental model to be used in spinal cord reconstruction studies. Function and anatomic results are undisputed only after spinal cord transection.

Animals↗
PubMed2006

Biomechanic study of the human liver during a frontal deceleration.

BACKGROUND: Mechanisms of hepatic injury remain poorly understood. Surgical literature reports some speculative theories that have never been proved. The aim of this study was to examine the behavior of the liver during brutal frontal deceleration. METHODS: Six trunks, removed from human cadavers, underwent free falls at 4, 6, and 8 meters per second (mps). Accelerometers were positioned in the two lobes of the liver, in front of the vertebra L2, and in the retro hepatic inferior vena cava. Relative motions of the lobes of the liver and of the two other anatomic marks were observed. In parallel, numerical simulations of this experiment have been performed using a finite element model. RESULTS: In the direction of impact, the vertebra L2 had no considerable displacement with the inferior vena cava. There was a noteworthy displacement between the two hepatic lobes. The left hepatic lobe had a large relative displacement with the vertebra L2 and the inferior vena cava. The right hepatic lobe was more stable with the vertebra L2 and the inferior vena cava. Numerical simulation of the same protocol underlined a rotation effect of the liver to the left around the axis of the inferior vena cava. CONCLUSIONS: These results support the surgical data. They highlight a crucial zone and explain how dramatic lacerations between the two lobes of the liver can occur.

Aged↗
PubMed2006

Influence of local environment on the differentiation of neural stem cells engrafted onto the injured spinal cord.

OBJECTIVES: In vitro, neural stem cells (NSCs) proliferate as undifferentiated spheroids and differentiate into neurons, astrocytes and oligodendrocytes. These features make NSCs suitable for spinal cord (SC) reconstruction. However, in vivo experiments have demonstrated that in the injured SC transplanted NSCs either remain undifferentiated or differentiate into the astrocytic phenotype. The microenvironment of the injured SC is believed to play a crucial role in driving the differentiation of the engrafted NSCs. Here, we tested the hypothesis that inflammatory cytokines (ICs) may be involved in the restricted differentiation of NSCs after grafting onto the injured SC. METHODS: As the first step, we used immunohistochemistry to analyse the expression of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and interferon (IFN)-gamma in the normal SC of mice and following traumatic injury. Then, we investigated whether a combination of TNF-alpha, IL-1beta and IFN-gamma may affect the phenotype of murine NSCs in vitro. RESULTS: We found that TNF-alpha, IL-1beta and IFN-gamma, which are absent in the normal SC, are all expressed in the injured SC and the expression of these cytokines follows a timely tuned fashion with IFN-gamma being detectable as long as 4 weeks after injury. In culture, exposure of proliferating NSCs to a combination of TNF-alpha, IL-1beta and IFN-gamma was per se sufficient to induce the astrocytic differentiation of these cells even in the absence of serum. CONCLUSIONS: In the traumatically injured SC, differentiation of engrafted NSCs is restricted towards the astrocytic lineage because of the inflammatory environment. ICs are likely to play a major role in differentiation of NSCs in the in vivo conditions.

Animals↗
PubMed2006

In vitro analysis of mouse neural stem cells genetically modified to stably express human NGF by a novel multigenic viral expression system.

OBJECTIVES: The purpose of this study was to characterize mouse neural stem cells (NSC) transduced by a multigenic lentiviral vector (LV) and stably express recombinant human nerve growth factor (rhNGF). We obtained NSC-derived cell lines which express human NGF in relevant amount to exploit their ability for therapeutic applications. METHODS: We constructed advanced multigenic LV vectors which contain a tricistronic cassette to express simultaneously up to three independent genes: (1) rhNGF (beta subunit); (2) EGFP (enhanced green fluorescent protein) and (3) Neo(R) (neomycin antibiotic resistance gene). Lentiviruses were obtained by transfecting LV constructs plus helper plasmids in human embryonic kidney (HEK)-293T packaging cells. Lentiviral virions were released in culture media and subsequent used to infect mouse NSC. Genetycin 418-resistant NSC were obtained after 1 month of selection in the presence of antibiotic (G418). Levels of human NGF secreted by rhNGF-NSC were determined by ELISA (enzyme-linked immunosorbent assay). Features of multipotentiality of engineered NSC-derived cell lines versus naive cells (control-NSC) were assessed by immunocytochemical analysis in differentiation conditions. Self-renewal of NSC was tested by neurospheres assay (NSA). RESULTS: Levels of secreted human NGF, from conditioned media obtained by rhNGF-NSC cultures, were found to be elevated in either proliferation or differentiation conditions as compared with control cells. Moreover, released hNGF demonstrated biologic activity on PC12 cells by a functional test of neurite outgrowth. Immunocytochemical analysis revealed that engineered NSC showed to be all positives for EGFP. After thirty passages in vitro in the presence of G418, engineered cells versus naive NSC cultures maintained their multipotentiality to differentiate into neurons, astrocytes and oligodendrocytes. Furthermore, it was found that rhNGF-NSC-derived neurons expressed choline acetyltransferase (ChAT) and displayed an enhanced axonal growth. NSA showed an altered sphere forming frequency either in rhNGF-NSC or both group of control NSC. DISCUSSION: Lentivirus-mediated rhNGF gene transfer into NSC was achieved using a new version of LV vectors. We obtained rhNGF-NSC-derived cell lines which released hNGF to high levels in the culture medium. The expression of neural differentiation markers, like microtubule associated protein 2 (MAP2) (a/b), glial fibrillary acidic protein (GFAP) and chondroitin sulphate proteoglycan (NG2), was not enhanced in rhNGF-NSC compared with control cells. Secreted hNGF increased axonal sprouting by rhNGF-NSC-derived neurons which was associated with ChAT expression. rhNGF-NSC may prospectively be good candidates for the treatment of either neurodegenerative diseases such as Alzheimer disease or central nervous system injuries.

Animals↗
PubMed2005

Transcriptional analysis of doxorubicin-induced cardiotoxicity.

Doxorubicin is an effective chemotherapeutic agent against a broad range of tumors. However, a threshold dose of doxorubicin causes an unacceptably high incidence of heart failure and limits its clinical utility. We have established two models of doxorubicin cardiotoxicity in mice: 1) in an acute model, mice are treated with 15 mg/kg of doxorubicin once; and 2) in a chronic model, they receive 3 mg/kg weekly for 12 wk. Using echocardiography, we have monitored left ventricular function during treatment in the chronic model and seen the expected development of dilated cardiomyopathy. Treated mice showed histological abnormalities similar to those seen in patients with doxorubicin cardiomyopathy. To investigate transcriptional regulation in these models, we used a muscle-specific cDNA microarray. We have identified genes that respond to doxorubicin exposure in both models and confirmed these results using real-time PCR. In the acute model, a set of genes is regulated early and rapidly returns to baseline levels, consistent with the half-life of doxorubicin. In the chronic model, which mimics the clinical situation much more closely, we identified dysregulated genes that implicate specific mechanisms of cardiac toxicity. These include STARS, a hypertrophy-responsive gene; SNF1-kinase, a potential modulator of ATP levels; and AXUD1, a downstream target of the proapoptotic regulator AXIN1.

Animals↗
PubMed2005

DATA-MEAns: an open source tool for the classification and management of neural ensemble recordings.

The number of laboratories using techniques that allow to acquire simultaneous recordings of as many units as possible is considerably increasing. However, the development of tools used to analyse this multi-neuronal activity is generally lagging behind the development of the tools used to acquire these data. Moreover, the data exchange between research groups using different multielectrode acquisition systems is hindered by commercial constraints such as exclusive file structures, high priced licenses and hard policies on intellectual rights. This paper presents a free open-source software for the classification and management of neural ensemble data. The main goal is to provide a graphical user interface that links the experimental data to a basic set of routines for analysis, visualization and classification in a consistent framework. To facilitate the adaptation and extension as well as the addition of new routines, tools and algorithms for data analysis, the source code and documentation are freely available.

Action Potentials↗
PubMed2005

Long survival in an untreated solitary choroid plexus metastasis from renal cell carcinoma: case report and review of the literature.

Brain metastases from renal cell carcinoma (RCC) are rare. Among them, the metastases localized only in the choroid plexus are exceptional and only six cases are reported in the literature. Here we report on a patient with a single choroid plexus metastasis from RCC which presented an unusual biological behaviour. For several years, such metastasis was interpreted as a benign intraventricular tumor and was not treated. Four years after the initial neuroradiological evidence, because of the appearance of symptoms, the brain metastasis was excised. We think that this unusual biological behaviour of the tumor determined the late inset of the neurological symptoms, despite the location at the choroid plexus that usually leads to an early obstructive hydrocephalus. To our knowledge, this 46 months survival is the longest survival of a patient affected by a single choroid plexus metastasis from RCC.

Aged↗
PubMed2005

Intraoperative microvascular Doppler in intracranial aneurysm surgery.

OBJECTIVE: The usefulness of intraoperative microvascular Doppler (IMD) in preventing an incorrect placement of the clip during intracranial aneurysm surgery is described. Such incorrect placement of the clip may cause new bleeding or ischemic accident as a consequence of incomplete exclusion of the aneurysm or stenosis/occlusion of the parent and/or adjacent arteries. METHODS: One hundred thirty patients, harboring 136 aneurysms, were operated on using IMD. IMD study was performed on the aneurysm and adjacent arterial vessels before and after the clip placement. Mechanical arterial spasm was treated by topical sodium nitroprusside (SNP) and its efficacy verified by IMD. RESULTS: In 55 aneurysms (42.3%), IMD was helpful in understanding the microvascular anatomy before clip placement. Complete exclusion resulted in 129 aneurysms (94.9%). In 5 cases (3.7%) in which IMD revealed a persistent blood flow in the aneurysm, the clip was repositioned. In 2 cases (1.4%), we obtained false-negative results. In 25 cases (18.3%) in which IMD data documented a severe flow reduction in the vessel harboring the aneurysm or in the vessel originating very close to the aneurysm neck, the clip was repositioned. There were no complications due to the use of IMD. In 20 patients (15%) in which IMD revealed arterial spasm by surgical manipulation, topical SNP was followed by resolution of the spasm. CONCLUSION: IMD is a feasible, safe, and very reliable technique in aneurysm surgery. Compared to other procedures such as intraoperative angiography, the cost efficiency of IMD is favorable.

Adolescent↗
PubMed2005

Homologous transplantation of neural stem cells to the injured spinal cord of mice.

OBJECTIVE: Murine neural stem cells (NSCs) were homografted onto the injured spinal cord (SC) to assess their potential to improve motor behavior, to differentiate as neurons, and to establish synapse-like contacts with the descending axonal paths of the host. In addition, we investigated whether transduced NSCs over-expressing vascular endothelial growth factor might exert any angiogenetic effect in the injured SC. METHODS: NSCs derived from mouse embryos were transduced to express either green fluorescent protein or vascular endothelial growth factor. The cells were engrafted in mice where an extended dorsal funiculotomy had been performed at the T8-T9 level. At intervals from 4 to 12 weeks after grafting, motor behavior was assessed using an open field locomotor scale and footprint analysis. At the same time points, the SC was studied by conventional histology, immunohistochemistry, and fluorescence microscopy. The interactions between the grafted NSCs and descending axonal paths were investigated using anterogradely transported fluorescent axonal tracers. RESULTS: By the 12-week time point, mice engrafted with NSCs significantly improved both their locomotor score on open field test and their base of support on footprint analysis. Histological studies showed that green fluorescent protein-positive NSCs survived as long as 12 weeks after grafting, migrated from the grafting site with a tropism toward the lesion, and either remained undifferentiated or differentiated into the astrocytic phenotype without neuronal or oligodendrocytic differentiation. Interestingly, the NSC-derived astrocytes expressed vimentin, suggesting that these cells differentiated as immature astrocytes. The tips of severed descending axonal paths came adjacent to grafted NSCs without forming synapse-like structures. When genetically engineered to over-express vascular endothelial growth factor, the grafted NSCs significantly increased vessel density in the injured area. CONCLUSION: In the traumatically injured mice SC, NSC grafting improves motor recovery. Although differentiation of engrafted NSCs is restricted exclusively toward the astrocytic phenotype, the NSC-derived astrocytes show features that are typical of the early phase after SC injury when the glial scar is still permissive to regenerating axons. The immature phenotype of the NSC-derived astrocytes suggests that these cells might support neurite outgrowth by the host neurons. Thus, modifying the glial scar with NSCs might enhance axonal regeneration in the injured area. The use of genetically engineered NSCs that express trophic factors appears to be an attractive tool in SC transplantation research.

Animals↗
PubMed2005

Pyrogenic cytokine interleukin-6 expression by a chordoid meningioma in an adult with a systemic inflammatory syndrome. Case report and review of the literature.

Chordoid meningioma is a rare meningothelial tumor characterized by chordoma-like histological features with lymphoplasmacellular infiltration. This tumor is often seen in children, but not in adults, with a systemic inflammatory syndrome (iron-resistant microcytic anemia and/or dysgammaglobulinemia) and very rarely with a persistent moderate hyperthermia. In the present report the authors describe a temporal chordoid meningioma in a 30-year-old woman who presented with fever, headache, and a serological inflammatory syndrome. The clinical symptomatology, chiefly the fever, disappeared immediately after removal of the tumor. To the authors' knowledge, only one similar patient with such clinical presentation and response to surgery has been mentioned in the literature. Interestingly, at immunohistochemical examination, the neoplasm showed focal positivity for the pyrogenic cytokine interleukin-6. The capacity of the tumor to produce this pyrogenic cytokine could explain both the patient's clinical presentation and her response to the surgical management.

Adult↗
PubMed2004

Long-term stimulation and recording with a penetrating microelectrode array in cat sciatic nerve.

We studied the consequences of long-term implantation of a penetrating microelectrode array in peripheral nerve over the time course of 4-6 mo. Electrode arrays without lead wires were implanted to test the ability of different containment systems to protect the array and nerve during contractions of surrounding muscles. Treadmill walking was monitored and the animals showed no functional deficits as a result of implantation. In a different set of experiments, electrodes with lead wires were implanted for up to 7 mo and the animals were tested at 2-4 week intervals at which time stimulation thresholds and recorded sensory activity were monitored for every electrode. It was shown that surgical technique highly affected the long-term stimulation results. Results between measurement sessions were compared, and in the best case, the stimulation properties stabilized in 80% of the electrodes over the course of the experiment (162 days). The recorded sensory signals, however, were not stable over time. A histological analysis performed on all implanted tissues indicated that the morphology and fiber density of the nerve around the electrodes were normal.

Action Potentials↗
PubMed2004

Motoneurons innervating facial muscles after hypoglossal and hemihypoglossal-facial nerve anastomosis in rats.

Hypoglossal-facial nerve anastomosis (HFA) is the most popular surgical procedure to reinnervate facial muscles after injury of the facial nerve. Section of the hypoglossus causes paralysis and atrophy of the hemi-tongue. In the attempt to overcome this consequence, the hemihypoglossal-facial nerve anastomosis (HHFA) has been proposed and only a half of the main trunk of the hypoglossus is connected to the distal stump of the facial nerve. In the rat, we have studied experimentally the anatomical nuclear changes after HFA and HHFA with the aim of establishing the quantitative motoneuron innervation of facial muscles obtained with each one of the two operative options. Horseradish peroxidase (HRP) injected in both types of anastomosis labeled not only hypoglossal motoneurons, but also facial motoneurons. HFA appeared to offer a significant quantitative motoneuron innervation higher than HHFA and then a higher probable better functional recovery. Both HFA and HHFA performed immediately after section of the facial nerve in rats did not result in a phenomenon of motor hyperinnervation. In our experimental model, the proximal facial nerve stump was coagulated at the stylomastoid foramen to avoid regeneration. Then, the labeled motoneurons into the facial nucleus could really be the expression of axonal projections from facial motoneurons to the hypoglossus nerve and facial muscles. No labeled motoneurons were seen contralaterally as we observed previously after section and repair of several nerves.

Anastomosis, Surgical↗
PubMed2004

Combined treatment of advanced stages of recurrent skin cancer of the head.

OBJECT: The authors investigated whether skull base resection and primary free-flap reconstruction in a single-stage surgery is oncologically effective for treating advanced stages of recurrent skin cancer (RSC) of the head. METHODS: Eighteen consecutive patients were surgically treated. Twelve of them underwent an anterolateral skull base resection, which was performed using a pterional craniotomy combined with an orbitozygomatic osteotomy. Six patients underwent a posterolateral skull base resection, which was performed using an asterional craniotomy combined with a retrolabyrinthine petrosectomy. The wide postoperative defects were covered with muscular or myocutaneous free flaps. The main factor influencing survival was the extent of the resection: patients with no or minimal residual disease showed a statistically significant longer survival time than those with consistent residual disease. Basal cell carcinoma had a better prognosis than squamous cell carcinoma. A trend toward improved survival was observed in patients classified as T4M0 with negative lymph nodes (N0), but this trend was not statistically significant. Adjuvant radiotherapy significantly influenced both survival time and the rate of local recurrence. The surgical morbidity rate was 27.8%; there were two transient cerebrospinal fluid leaks and three seventh cranial nerve injuries. Late complications included radionecrosis in one patient and skin erosion requiring a second surgery in another patient. No deaths occurred during a 30-day postoperative period. CONCLUSIONS: Advances in skull base surgery and free-flap reconstruction allowed the authors to treat patients with advanced-stage RSC of the head in a rather satisfactory manner. Only when it is impossible to achieve no or minimal residual disease should aggressive treatment be considered.

Aged↗
PubMed2003

Reinnervation of extraocular muscles by facial-to-oculomotor nerve anastomosis in rats: anatomic nuclear changes.

OBJECTIVE: Oculomotor nerve palsy greatly impairs the patient's daily life. After oculomotor nerve injury, when the central nerve stump is not available, neurotization of the distal nerve stump with a donor nerve may be performed. Here, we present an experimental anatomic study in rats related to the motor nuclear organization after facial-to-oculomotor nerve anastomosis. METHODS: In adult rats, the right oculomotor nerve was transected at the skull base. Then, the ipsilateral facial nerve was exposed at the stylomastoid foramen and connected side-to-end to one extremity of a peroneal nerve autograft. The other extremity of the nerve autograft was connected end-to-end to the distal stump of the transected oculomotor nerve. Twelve weeks later, axonal regeneration in the autograft and brainstem somatotopic representation of the reinnervated extraocular muscles were investigated by use of histological and retrograde axonal tracing techniques. RESULTS: The autograft was reinnervated by a large number of small axons, 1 to 5 microm in diameter. After tracer injection into the superior rectus and medial rectus muscles, retrogradely labeled neurons were seen not only in the ipsilateral facial nucleus (16%) but also in the contralateral nucleus (8%). Labeled neurons were also seen in the ipsilateral abducens (12%), motor trigeminus (7%), trochlear (23%), and contralateral trochlear (34%) nuclei. In normal rats, the extraocular muscles are innervated by unilateral-ipsilateral brainstem motor nuclei, except for the superior rectus and superior oblique muscles, which are innervated by bilateral, primarily contralateral, nuclei. CONCLUSION: The central rearrangement of the extraocular muscle nuclei after facial-to-oculomotor nerve anastomosis represents an original example of plasticity. Functional studies are needed to demonstrate whether this procedure might serve to restore some degree of eye motility.

Anastomosis, Surgical↗
PubMed2003

Chordoma of the skull base: predictors of tumor recurrence.

OBJECT: Chordomas of the skull base are generally regarded as slow-growing tumors; however, approximately 20% of these lesions have been shown to recur as early as 1 year postsurgery. The classic pathological paradigms are poor predictors of outcome, and additional markers are needed to identify patients at risk for early tumor recurrence. In this study the authors describe such a marker. METHODS: In a series of 26 patients with chordomas of the skull base, the authors investigated the relationship between the biological behavior of the tumor, which was determined according to the interval for its recurrence and volume doubling time, and several pathological and molecular features, which included the histological variant, proliferative activity, mutation of p53 protein, expression of human telomerase reverse transcriptase (hTERT) messenger (m)RNA, loss of heterozygosity (LOH), and microsatellite instability. The major finding in this study was that hTERT mRNA expression in chordoma cells identifies those tumors that exhibit unusually fast rates of growth. The expression of hTERT mRNA was frequently associated with mutation of p53 protein, indicating that telomerase dysfunction combines with abnormal p53 function to initiate the unrestrained clonal expansion of the tumor cells. In cases in which the tumor was partially removed, mutation of p53 protein and expression of hTERT mRNA predicted increased doubling time for residual tumor as well as the probability of tumor recurrence. Cell proliferation, as investigated using the Ki-67 method, was significantly related to the tumor doubling time; however, the authors found that the pattern of cell proliferation was not homogeneous throughout the chordoma tissue, and that the proliferative index might change by a factor as high as 8 among different regions of the same tumor. The LOH and microsatellite instability do not seem to affect the prognosis of skull base chordomas. CONCLUSIONS: Reactivation of telomerase in chordomas is a reliable predictor of outcome. The ability to predict the biological behavior of chordomas might have immediate implications in the management of this disease in patients who undergo surgery.

Adult↗