Therapeutic potential of human olfactory bulb neural stem cells for spinal cord injury in rats.

STUDY DESIGN: Adult human olfactory bulb neural stem cells (OBNSCs) were isolated from human patients undergoing craniotomy for tumor resection. They were genetically engineered to overexpresses green fluorescent protein (GFP) to help trace them following engraftment. Spinal cord injury (SCI) was induced in rats using standard laminectomy protocol, and GFP-OBNSC were engrafted into rat model of SCI at day 7 post injury. Three rat groups were used: (i) Control group, (ii) Sham group (injected with cerebrospinal fluid) and treated group (engrafted with OBNSCs). Tissues from different groups were collected weekly up to 2 months. The collected tissues were fixed in 4% paraformaldehyde, processed for paraffin sectioning, immunohistochemically stained for different neuronal and glial markers and examined with bright-field fluorescent microscopy. Restoration of sensory motor functions we assessed on a weekly bases using the BBB score. OBJECTIVES: To assess the therapeutic potential of OBNSCs-GFP and their ability to survive, proliferate, differentiate and to restore lost sensory motor functions following their engraftment in spinal cord injury (SCI). METHODS: GFP-OBNSC were engrafted into a rat model of SCI at day 7 post injury and were followed-up to 8 weeks using behavioral and histochemical methods. RESULTS: All transplanted animals exhibited successful engraftment. The survival rate was about 30% of initially transplanted cells. Twenty-seven percent of the engrafted cells differentiated along the NG2 and O4-positive oligodendrocyte lineage, 16% into MAP2 and ß-tubulin-positive neurons, and 56% into GFAP-positive astrocytes. CONCLUSION: GFP-OBNSCs had survived for >8 weeks after engraftment and were differentiated into neurons, astrocytes and oligodendrocytes, The engrafted cells were distributed throughout gray and white matter of the cord with no evidence of abnormal morphology or any mass formation indicative of tumorigenesis. However, the engrafted cells failed to restore lost sensory and motor functions as evident from behavioral analysis using the BBB score test.

Mesenchymal differentiation of glioblastoma stem cells.

Glioblastoma multiforme is a severe form of cancer most likely arising from the transformation of stem or progenitor cells resident in the brain. Although the tumorigenic population in glioblastoma is defined as composed by cancer stem cells (CSCs), the cellular target of the transformation hit remains to be identified. Glioma stem cells (SCs) are thought to have a differentiation potential restricted to the neural lineage. However, using orthotopic versus heterotopic xenograft models and in vitro differentiation assays, we found that a subset of glioblastomas contained CSCs with both neural and mesenchymal potential. Subcutaneous injection of CSCs or single CSC clones from two of seven patients produced tumor xenografts containing osteo-chondrogenic areas in the context of glioblastoma-like tumor lesions. Moreover, CSC clones from four of seven cases generated both neural and chondrogenic cells in vitro. Interestingly, mesenchymal differentiation of the tumor xenografts was associated with reduction of both growth rate and mitotic index. These findings suggest that in a subclass of glioblastomas the tumorigenic hit occurs on a multipotent stem cell, which may reveal its plasticity under specific environmental stimuli. The discovery of such biological properties might provide considerable information to the development of new therapeutic strategies aimed at forcing glioblastoma stem cell differentiation.

G-quadruplex ligand RHPS4 radiosensitizes glioblastoma xenograft in vivo through a differential targeting of bulky differentiated- and stem-cancer cells.

BACKGROUND: Glioblastoma is the most aggressive and most lethal primary brain tumor in the adulthood. Current standard therapies are not curative and novel therapeutic options are urgently required. Present knowledge suggests that the continued glioblastoma growth and recurrence is determined by glioblastoma stem-like cells (GSCs), which display self-renewal, tumorigenic potential, and increased radio- and chemo-resistance. The G-quadruplex ligand RHPS4 displays in vitro radiosensitizing effect in GBM radioresistant cells through the targeting and dysfunctionalization of telomeres but RHPS4 and Ionizing Radiation (IR) combined treatment efficacy in vivo has not been explored so far. METHODS: RHPS4 and IR combined effects were tested in vivo in a heterotopic mice xenograft model and in vitro in stem-like cells derived from U251MG and from four GBM patients. Cell growth assays, cytogenetic analysis, immunoblotting, gene expression and cytofluorimetric analysis were performed in order to characterize the response of differentiated and stem-like cells to RHPS4 and IR in single and combined treatments. RESULTS: RHPS4 administration and IR exposure is very effective in blocking tumor growth in vivo up to 65 days. The tumor volume reduction and the long-term tumor control suggested the targeting of the stem cell compartment. Interestingly, RHPS4 treatment was able to strongly reduce cell proliferation in GSCs but, unexpectedly, did not synergize with IR. Lack of radiosensitization was supported by the GSCs telomeric-resistance observed as the total absence of telomere-involving chromosomal aberrations. Remarkably, RHPS4 treatment determined a strong reduction of CHK1 and RAD51 proteins and transcript levels suggesting that the inhibition of GSCs growth is determined by the impairment of the replication stress (RS) response and DNA repair. CONCLUSIONS: We propose that the potent antiproliferative effect of RHPS4 in GSCs is not determined by telomeric dysfunction but is achieved by the induction of RS and by the concomitant depletion of CHK1 and RAD51, leading to DNA damage and cell death. These data open to novel therapeutic options for the targeting of GSCs, indicating that the combined inhibition of cell-cycle checkpoints and DNA repair proteins provides the most effective means to overcome resistance of GSC to genotoxic insults.

Type-3 metabotropic glutamate receptors negatively modulate bone morphogenetic protein receptor signaling and support the tumourigenic potential of glioma-initiating cells.

Targeted-therapies enhancing differentiation of glioma-initiating cells (GICs) are potential innovative approaches to the treatment of malignant gliomas. These cells support tumour growth and recurrence and are resistant to radiotherapy and chemotherapy. We have found that GICs express mGlu3 metabotropic glutamate receptors. Activation of these receptors sustained the undifferentiated state of GICs in culture by negatively modulating the action of bone morphogenetic proteins, which physiologically signal through the phosphorylation of the transcription factors, Smads. The cross-talk between mGlu3 receptors and BMP receptors was mediated by the activation of the mitogen-activated protein kinase pathway. Remarkably, pharmacological blockade of mGlu3 receptors stimulated the differentiation of cultured GICs into astrocytes, an effect that appeared to be long lasting, independent of the growth conditions, and irreversible. In in vivo experiments, a 3-month treatment with the brain-permeant mGlu receptor antagonist, LY341495 limited the growth of infiltrating brain tumours originating from GICs implanted into the brain parenchyma of nude mice. While clusters of tumour cells were consistently found in the brain of control mice, they were virtually absent in a large proportion of mice treated with LY341495. These findings pave the way to a new non-cytotoxic treatment of malignant gliomas based on the use of mGlu3 receptor antagonists.

Primary hemorrhagic intramedullary melanoma. Case report with emphasis on the difficult preoperative diagnosis.

Primary melanoma of the central nervous system (CNS) is rare and primary spinal melanoma (PSM) is even more unusual. Preoperative diagnosis of melanocytic lesion as a PSM is difficult, because of the heterogeneous magnetic resonance (MR) signal intensity, due to hemorrhagic foci and melanin deposits. We describe the case of a 68 year-old male with a MR showing at Th8-Th9 level a well-defined intramedullary lesion; for the characteristics of hemorrhagic signal on MR and its association with a presumptive brain cavernoma, a preoperative diagnosis of intramedullary cavernous angioma was suspected. Pathological examination revealed a melanoma, and for the absence of other localizations outside the spinal cord, a diagnosis of primary spinal melanoma was established. The growth of PSM is slower and survival is longer than in the most common spinal metastasis from skin melanoma. Patients who undergo surgical excision, alone or associated with additional treatments, often show a long survival. We report this case to underline the importance and difficulties concerning the preoperative diagnosis of a hemorrhagic intramedullary lesion.

Gadolinium in human glioblastoma cells for gadolinium neutron capture therapy.

157Gd is a potential agent for neutron capture cancer therapy (GdNCT). We directly observed the microdistribution of Gd in cultured human glioblastoma cells exposed to Gd-diethylenetriaminepentaacetic acid (Gd-DTPA). We demonstrated, with three independent techniques, that Gd-DTPA penetrates the plasma membrane, and we observed no deleterious effect on cell survival. A systematic microchemical analysis revealed a higher Gd accumulation in cell nuclei compared with cytoplasm. This is significant for prospective GdNCT because the proximity of Gd to DNA increases the cell-killing potential of the short-range, high-energy electrons emitted during the neutron capture reaction. We also exposed Gd-containing cells to thermal neutrons and demonstrated the GdNC reaction effectiveness in inducing cell death. These results in vitro stimulated in vivo Gd-DTPA uptake studies, currently underway, in human glioblastoma patients.

Metabolic/Proteomic Signature Defines Two Glioblastoma Subtypes With Different Clinical Outcome.

Glioblastoma (GBM) is one of the deadliest human cancers. Because of the extremely unfavorable prognosis of GBM, it is important to develop more effective diagnostic and therapeutic strategies based on biologically and clinically relevant subclassification systems. Analyzing a collection of seventeen patient-derived glioblastoma stem-like cells (GSCs) by gene expression profiling, NMR spectroscopy and signal transduction pathway activation, we identified two GSC clusters, one characterized by a pro-neural-like phenotype and the other showing a mesenchymal-like phenotype. Evaluating the levels of proteins differentially expressed by the two GSC clusters in the TCGA GBM sample collection, we found that SRC activation is associated with a GBM subgroup showing better prognosis whereas activation of RPS6, an effector of mTOR pathway, identifies a subgroup with a worse prognosis. The two clusters are also differentiated by NMR spectroscopy profiles suggesting a potential prognostic stratification based on metabolic evaluation. Our data show that the metabolic/proteomic profile of GSCs is informative of the genomic/proteomic GBM landscape, which differs among tumor subtypes and is associated with clinical outcome.

Glioblastoma stem cells: radiobiological response to ionising radiations of different qualities.

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumour, with very poor prognosis. The high recurrence rate and failure of conventional treatments are expected to be related to the presence of radio-resistant cancer stem cells (CSCs) inside the tumour mass. CSCs can both self-renew and differentiate into the heterogeneous lineages of cancer cells. Recent evidence showed a higher effectiveness of C-ions and protons in inactivating CSCs, suggesting a potential advantage of Hadrontherapy compared with conventional radiotherapy for GBM treatment. To investigate the mechanisms involved in the molecular and cellular responses of CSCs to ionising radiations, two GBM stem cell (GSC) lines, named lines 1 and 83, which were derived from patients with different clinical outcomes and having different metabolic profiles (as shown by NMR spectroscopy), were irradiated with (137)Cs photons and with protons or C-ions of 62 MeV u(-1) in the dose range of 5-40 Gy. The biological effects investigated were: cell death, cell cycle progression, and DNA damage induction and repair. Preliminary results show a different response to ionising radiation between the two GSC lines for the different end points investigated. Further experiments are in progress to consolidate the data and to get more insights on the influence of radiation quality.

Normal rhodanese activity in leukocytes from Leber patients: enzyme characterization and activity levels.

The thiosulfate:cyanide sulfurtransferase (rhodanese) enzyme (EC 2.8.1.1.) was studied in human leukocytes from control subjects and from nine patients with Leber's hereditary optic atrophy. Enzyme activity was proportional to protein concentration in the tested range (0.09 to 0.39 mg) as well as to incubation time. The optimal pH for reaction was 8.7 and the apparent Km for thiosulfate was 7 X 10(-3) M. No significant difference of enzyme activity was present in Leber's disease.

Telomerase expression in intracranial tumours: prognostic potential for malignant gliomas and meningiomas.

AIM: To evaluate the diagnostic value of telomerase expression in intracranial tumours. METHODS: 98 surgical specimens from different neoplasms were analysed by the telomeric repeat amplification protocol (TRAP) and the presence of telomerase compared with the histological diagnosis and the proliferation index. RESULTS: A high degree of positivity for telomerase activity was found in glioblastomas and atypical/anaplastic meningiomas. Telomerase activity was poorly detected in anaplastic astrocytomas. CONCLUSIONS: The TRAP assay seems to be a valuable index for identifying meningeal tumours with aggressive behaviour.

Interhemispheric integration of simple visuomotor responses in patients with partial callosal defects.

Because of the organization of visual and motor pathways, simple manual responses to a light stimulus in the right or left visual hemifields are performed faster with uncrossed hand-field combinations than with crossed hand-field combinations. Uncrossed responses can be integrated within a single hemisphere, whereas crossed responses require a time-consuming interhemispheric transfer via the corpus callosum which is reflected in the difference between crossed and uncrossed reaction times. We investigated crossed-uncrossed differences (CUDs) in speed of simple visuomotor responses to lateralized flashes in seven subjects with an anterior section of the corpus callosum sparing the splenium and in one subject with an agenetic absence of the splenium due to a cerebrovascular malformation. There was no evidence of an abnormal prolongation of the CUDs in any of these subjects, in sharp contrast with the very long CUDs exhibited by an epileptic subject with a complete callosal section and two subjects with total callosal agenesis tested in the same experimental situation [1]. The normality of the CUDs in the subjects with partial callosal defects was not due to a postoperatory reorganization of interhemispheric communication, since there was no indication of an increased CUD in a patient tested as early as 5 days after the anterior callosotomy. These results are compatible with the assumption that both anterior and posterior callosal routes may subserve the integration of speeded manual responses to a visual stimulus directed to the hemisphere ipsilateral to the responding hand.

Effects of topically administered nerve growth factor on axonal regeneration in peripheral nerve autografts implanted in the spinal cord of rats.

The effect of exogenous nerve growth factor (NGF) on axonal regeneration into autologous peripheral nerve (PN) grafts implanted to the spinal cord (SC) of rats was assessed by retrograde labeling of the parent soma of the regenerating axons with horseradish peroxidase. NGF was delivered at the graft site over periods of 15 and 30 days by using indwelling osmotic minipumps. In control rats, the minipumps were filled with saline. At 15 days after grafting in the NGF-treated rats, the mean number of SC as well as dorsal root ganglion (DRG) neurons that regenerated their axons into the peripheral nerve grafts was increased 55.3 and 26.4 times, respectively, as compared to the control group values. At 30 days, SC and DRG neurons in the NGF-treated group were 10.9 and 3.1 times greater than in the control group. In the NGF-treated group, the regenerating SC neurons were located within a range of 7 to 13 mm from the graft site as compared to 1 to 7 mm in the control group. Finally, the analysis of the soma diameters of the regenerating neurons showed that NGF enhanced and maintained with time the regenerative response from small-sized DRG neurons. Therefore, NGF is thought to promote directly the regenerative potential of SC as well as DRG neurons and to exert an indirect glial cell-mediated effect at the SC-graft interface.

Quantitative, morphological, and somatotopic nuclear changes after facial nerve regeneration in adult rats: a possible challenge to the "no new neurons" dogma.

The anatomic reorganization of the subnucleus that controls the stylohyoid muscle (the stylohyoid subnucleus) within the brain stem facial nucleus was studied after regeneration of the facial nerve in adult rats. Horseradish peroxidase was injected into the right stylohyoid muscle 3 to 21 months after transection and repair of the right facial nerve at the level of the stylomastoid foramen. Position, number, and soma diameter of retrogradely horseradish peroxidase-labeled motoneurons were established, as well as the rostro-caudal extension of the stylohyoid subnucleus. In experimental rats, the stylohyoid subnucleus showed either an ipsilateral (50% of the rats) or a bilateral representation. In all of the experimental rats, the motoneurons composing the stylohyoid subnucleus had a more dispersed horizontal distribution pattern when compared with controls. More than 80% of the motoneurons were located outside the borders of the normal stylohyoid subnucleus, either ventrally or, especially in the rostral sections, dorsally closer to the floor of the fourth ventricle. The mean rostro-caudal length of the stylohyoid subnucleus was 2028.6 +/- 152.7 microns. The mean motoneuron number was 481.4 +/- 109.5 (2.20-fold greater than control values), and the motoneuron diameter distribution ranged from 7 to 43 microns. This study demonstrates that after regeneration of the facial nerve in adult rats, major changes occur in both the location and number of motoneurons that make up the stylohyoid subnucleus.(ABSTRACT TRUNCATED AT 250 WORDS)

Spinal cord transection in adult rats: effects of local infusion of nerve growth factor on the corticospinal tract axons.

The spinal cord of adult female rats was completely transected at the T8 level. Nerve growth factor (NGF) was administered at the lesion site via indwelling, implanted, osmotic minipumps. Purified NGF was supplied at doses of 100, 200, and 500 micrograms during a 30-day period. Control rats were treated with saline. At the end of the treatment, the proximal stump of corticospinal tract axons in the spinal cord was labeled with anterograde transported horseradish peroxidase (HRP) injected into the sensorimotor cortex. In control rats, the corticospinal tract axons ended abruptly, proximal to the zone of maximal damage. Sterile swellings developed at the axon tips, and no labeled axonal sprouts were apparent. On the contrary, in NGF-treated animals, the leading front of the corticospinal tract axons showed a trend of approaching the zone of maximal damage following abnormal paths through the dorsal-injured white matter. Axonal sprouts were seen more proximally, traveling toward the transection site in aberrantly located dorsal paths, completely outside the normal position of the corticospinal tract. NGF seems to partly restore the pattern of the regenerative behavior of the severed corticospinal tract axons after spinal cord transection in newborn rats, i.e., the induction of axonal sprouting in aberrantly located dorsal paths. An automated image analysis of the HRP reaction field close to the transection site demonstrated that the density of HRP-labeled axons in the corticospinal tract was significantly higher in the NGF-treated rats than in the control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Reconstruction of peripheral nerves: the phenomenon of bilateral reinnervation of muscles originally innervated by unilateral motoneurons.

It is well known that after reconstruction of sectioned peripheral nerves in adult mammals, denervated muscles are reinnervated by the axotomized motoneurons lying in the original motonucleus. It is less well known that these muscles can also be reinnervated by uninjured motoneurons lying in the homologous contralateral motonucleus. Therefore, after nerve reconstruction, bilateral motoneuron reinnervation of muscles can occur. Contralateral motoneurons sprout axons that cross the midline, grow in the reconstructed nerve, and reach muscle targets. This phenomenon was observed after reconstruction of several different peripheral nerves in adult mammals, including the oculomotor nerve in guinea pigs and the facial and sciatic nerves in rats. The retrograde axonal transport of horseradish peroxidase was used for the study of the organization of the brainstem and spinal cord motonuclei. Horseradish peroxidase was injected into the medial rectus muscle, the stylohyoid muscle, and the trunk of the sciatic nerve. The distance between the homologous motonuclei of both sides influenced the occurrence of this phenomenon. In fact, bilateral reinnervation of muscles after nerve reconstruction was found in 36% (sciatic nerve), 50% (facial nerve), and 100% (oculomotor nerve) of the operated animals. The total number of contralateral motoneurons found were 14% (oculomotor nerve), 8% (facial nerve), and 5% (sciatic nerve). Bilateral reinnervation of muscles was evoked by both immediate and delayed peripheral nerve repair and was a stable phenomenon, seen between 3 and 21 months after facial nerve reconstruction.

Alternative to tarsorrhaphy in peripheral facial nerve palsy with expectation of functional recovery. Technical note.

A simple technique is described for protecting the cornea in patients with peripheral facial nerve palsy while waiting for recovery of nerve function. The application of an adhesive strip to the superior eyelid permits opening and closing of the eye, and provides good protection of the cornea.

Retrograde degeneration of corticospinal axons following transection of the spinal cord in rats. A quantitative study with anterogradely transported horseradish peroxidase.

The extent of the retrograde degeneration of corticospinal axons following transection of the spinal cord was studied in rats by labeling corticospinal axons with anterogradely transported horseradish peroxidase injected in the sensorimotor cortex. Axotomized corticospinal axons underwent progressive and continuing retrograde degeneration. In specimens examined 5, 14, 28, and 56 days after trauma, the tips of the transected corticospinal axons were seen to terminate at 181 +/- 80 microns, 977 +/- 203 microns, 1751 +/- 344 microns, and 2559 +/- 466 microns (mean +/- standard deviation), respectively, from the site of transection. The rate of retrograde degeneration varied according to the interval after spinal cord transection, as follows: 36.2 microns/day during the first 5 days; 88.4 microns/day between 5 and 14 days; 55.3 microns/day between 14 and 28 days; and 28.8 microns/day between 28 and 56 days. These findings may serve as useful parameters for the objective assessment of therapeutic modalities in spinal cord injury research.

Sleep palsy (Saturday-night palsy) of the deep radial nerve. Case report.

A patient with a long-recognized asymptomatic lipoma adjacent to the deep radial nerve developed paralysis of this nerve from a compression similar to the sleep palsy, or "Saturday-night palsy," mechanism.