Onscreen-guided resection of extra-axial and intra-axial forebrain masses through registration of a variable-suction tissue resection device with a neuronavigation system.

OBJECTIVES: To describe a novel surgical technique in which neuronavigation is used to guide a tissue resection device during excision of forebrain masses in locations difficult to visualize optically. STUDY DESIGN: Short case series. ANIMALS: Six dogs and one cat with forebrain masses (five neoplastic, two nonneoplastic) undergoing excision with a novel tissue resection device and veterinary neuronavigation system. METHODS: The animals and resection instrument were coregistered to the neuronavigation system. Surgery was guided by real-time onscreen visualization of the resection instrument position relative to the preoperative MR images. Surgical outcome was evaluated by calculating residual tumor volume according to postoperative MRI. RESULTS: The technique was technically simple and led to the collection of diagnostic tissue samples in all cases. Postoperative MRI was available in six cases, two with gross-total resection, three with near-total resection, and one with subtotal resection. CONCLUSION: Neuronavigation-guided resection of intra-axial and extra-axial brain masses with the resection device resulted in gross-total or near-total resection in five of six animals with tumors otherwise difficult to visualize. Risk of brain shift limited absolute reliance on navigation images. CLINICAL SIGNIFICANCE: Real-time neuronavigation assistance is a feasible method for guidance and successful resection of brain masses that are poorly visualized because of intra-axial or deep location, tumor appearance, or hemorrhage.

Adipofascial Radial Forearm Free Flap for Anterior Skull Base Reconstruction in Complicated Forebrain Oncological Surgery.

BACKGROUND: Radical resections of ethmoidal tumors with intracranial extension present highly complex surgical and reconstructive problems. The purpose of report is to describe the authors' use of adipofascial radial forearm free flaps following unsuccessful anterior cranial fossa oncological surgery. METHODS: Adipofascial radial forearm free flaps were used to treat 3 similar cases of cutaneous fistula following bone resorption with communication to anterior cranial fossa and nasal cavity. RESULTS: No flap loss, no deaths, and no postoperative complications were observed. All patients underwent a nasal endoscopy, revealing adequate vitality and integration of the free flaps. One of the patients consented to additional surgery to improve outcome. CONCLUSIONS: Meticulous preoperative selection and an experienced interdisciplinary team are required to achieve the best surgical outcomes in complex cases. Free adipofascial forearm flaps could be an excellent therapeutic option in the reconstruction of the anterior skull base, notably in cases involving major postoperative complications.

[Surgical anatomy of the peri-insular association tracts. Part I.The superior longitudinal fascicle system]. / Khirurgicheskaya anatomiya periinsulyarnykh assotsiativnykh provodyashchikh putei. Chast' I. Sistema verkhnego prodol'nogo puchka.

AIM: To study the peri-insular association tract anatomy and define the permissible anatomical boundaries for resection of glial insular tumors with allowance for the surgical anatomy of the peri-insular association tracts. MATERIAL AND METHODS: In an anatomic study of the superior longitudinal fascicle system (SLF I, SLF II, SLF III, arcuate fascicle), we used 12 anatomical specimens (6 left and 6 right hemispheres) prepared according to the Klingler's fiber dissection technique. To confirm the dissection data, we used MR tractography (HARDI-CSD-tractography) of the conduction tracts, which was performed in two healthy volunteers. RESULTS: Except the SLF I (identified in 7 hemispheres by fiber dissection), all fascicles of the SLF system were found in all investigated hemispheres by both fiber dissection and MR tractography. The transcortical approach to the insula through the frontal and (or) parietal operculum is associated with a significant risk of transverse transection of the SLF III fibers passing in the frontal and parietal opercula. The most optimal area for the transcortical approach to the insula is the anterior third of the superior temporal gyrus that lacks important association tracts and, consequently, a risk of their injury. The superior peri-insular sulcus is an intraoperative landmark for the transsylvian approach, which enables identification of the SLF II and arcuate fascicle in the surgical wound. CONCLUSION: Detailed knowledge of the peri-insular association tract anatomy is the prerequisite for neurosurgery in the insular region. Our findings facilitate correct identification of both the site for cerebral operculum dissection upon the transcortical approach and the intraoperative landmarks for locating the association tracts in the surgical wound upon the transsylvian approach to the insula.

[Lateral differences in the forebrain and midbrain control of learned vocalizations in adult male Zebra Finch (Taeniopygia guttata)].

Learned vocalizations (long call and song) of adult male songbirds start from the high vocal center (HVC), and are integrated and output by the robust nucleus of the arcopallium (RA), which connects synaptic relationships with the dorsomedial nucleus of the intercollicular complex (DM). To determine the effect on learned vocalization of the unilateral forebrain and midbrain in adult male zebra finch, electrolytic lesions and acoustic analysis technology were used. The results indicated that RA and DM nuclei are involved in the control of learned vocalization, and the right side is dominant in the forebrain and midbrain.

Time-lapse imaging of neuroblast migration in acute slices of the adult mouse forebrain.

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB). In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes using blood vessels as a structural support and a source of molecular factors required for migration. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network. In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

Ultrasound-guided microinjection into the mouse forebrain in utero at E9.5.

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

2-Deoxy-D-glucose, but not mercaptoacetate, increases food intake in decerebrate rats.

We examined food intake in chronically maintained decerebrate rats in response to two antimetabolic drugs known to stimulate food intake, 2-mercaptoacetate (MA) and 2-deoxy-D-glucose (2DG). MA reduces fatty acid oxidation, and 2DG reduces glucose utilization. Because previous work has shown that insulin-induced hypoglycemia increases food intake in decerebrate rats, we predicted that 2DG would have this same effect. MA-induced feeding requires vagal sensory neurons that terminate in the hindbrain. Cholecystokinin-induced suppression of feeding, which likewise requires vagal sensory neurons, has been shown to suppress food intake in decerebrate rats. Therefore, we predicted that MA's effects on feeding would also persist in decerebrate rats. In our experiments, the test diet (40% milk, diluted with water) was infused intraorally through a chronic cheek fistula. We found that sham controls consumed 258% and 230% of their baseline milk intake in response to 2DG and MA, respectively. Decerebrates consumed 239% of their baseline milk intake in response to 2DG, but did not increase their intake in response to MA. Because decerebration separates the hindbrain from the forebrain, these results indicate that 2DG-induced glucoprivation is capable of acting within the hindbrain to activate fundamental reflex circuitry for consummatory feeding responses, as shown previously for hypoglycemia. In contrast, MA affects food consumption only after forebrain processing of MA-induced vagal afferent signals and in the presence of intact ascending and descending neural pathways.

Effects of gamma knife irradiation on the expression of NMDA receptor subunits in rat forebrain.

OBJECTIVE: To examine the effects of gamma knife surgery (GKS) on the expression of N-methel-D-asparate receptor (NMDAR) subunits in rat forebrain. MATERIALS AND METHODS: Using stereotactic technique, we performed gamma knife irradiation on the left forebrain of 13 male Wistar rats with a maximum dose of 60 Gy. These animals were raised for 24h, 30 and 60 days before they were killed. Then immunohistochemistry was applied to detect the relative levels of NMDAR subunits (NR1, NR2A, and NR2B) in the target region. RESULTS: The expression of NR1 and NR2A but not NR2B increased significantly in the cortex 30 and 60 days after irradiation. However, no significant differences in the expression of these three subunits were detected in the caudate putamen at all time points. CONCLUSION: gamma knife irradiation induced the upregulation of NMDAR subunits, NR1, and NR2A, which might represent a possible mechanism underlying the therapeutic effects of gamma knife irradiation on many neurological diseases, including drug resistance epilepsy.

Culture of mouse neural stem cell precursors.

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types. This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

Transplanted human embryonic germ cell-derived neural stem cells replace neurons and oligodendrocytes in the forebrain of neonatal mice with excitotoxic brain damage.

Stem cell therapy is a hope for the treatment of some childhood neurological disorders. We examined whether human neural stem cells (hNSCs) replace lost cells in a newborn mouse model of brain damage. Excitotoxic lesions were made in neonatal mouse forebrain with the N-methyl-D-aspartate (NMDA) receptor agonist quinolinic acid (QA). QA induced apoptosis in neocortex, hippocampus, striatum, white matter, and subventricular zone. This degeneration was associated with production of cleaved caspase-3. Cells immunopositive for inducible nitric oxide synthase were present in damaged white matter and subventricular zone. Three days after injury, mice received brain parenchymal or intraventricular injections of hNSCs derived from embryonic germ (EG) cells. Human cells were prelabeled in vitro with DiD for in vivo tracking. The locations of hNSCs within the mouse brain were determined through DiD fluorescence and immunodetection of human-specific nestin and nuclear antigen 7 days after transplantation. hNSCs survived transplantation into the lesioned mouse brain, as evidenced by human cell markers and DiD fluorescence. The cells migrated away from the injection site and were found at sites of injury within the striatum, hippocampus, thalamus, and white matter tracts and at remote locations in the brain. Subsets of grafted cells expressed neuronal and glial cell markers. hNSCs restored partially the complement of striatal neurons in brain-damaged mice. We conclude that human EG cell-derived NSCs can engraft successfully into injured newborn brain, where they can survive and disseminate into the lesioned areas, differentiate into neuronal and glial cells, and replace lost neurons. (c) 2005 Wiley-Liss, Inc.

Energy expenditure by intravenous administration of glucagon-like peptide-1 mediated by the lower brainstem and sympathoadrenal system.

Glucagon-like peptide-1 (GLP-1) is released from the gut in response to nutrient ingestion. Intravenous (iv) administration of GLP-1 (50 pmol-20 nmol) elicited dose-dependent increases in the rate of whole-body O2 consumption (VO2), an index of energy expenditure, and heart rate of urethane-anesthetized rats. The body core (colonic) temperature increased up to 0.3 degrees C without accompanying alteration of tail skin temperature. Intracerebroventricular (icv) administration of GLP-1 induced a slower and smaller increase in VO2 than the intravenous administration. The injection of glucagon-like peptide-2 (iv or icv) had no effect on VO2, body temperatures, or heart rate. Decerebration had no effect on the thermogenic responses induced by the iv administration of GLP-1, suggesting that the forebrain is not essential for these responses. However, cervical spinal transection greatly attenuated the responses, suggesting the critical involvement of the lower brainstem. Adrenalectomy or pretreatment with an autonomic ganglion blocker, hexamethonium, or a beta-adrenergic blocker, propranolol, also significantly attenuated the thermogenic response. However, subdiaphragmatic vagotomy or celiac-superior mesenteric ganglionectomy had no effect. Rats made insulin-deficient by pretreatment with streptozotocin also exhibited the normal thermogenic response to GLP-1. These results suggest the involvement of the GLP-1 in postprandial energy expenditure, mediated by the lower brainstem and sympathoadrenal system.

Transplantation of multipotent cells extracted from adult skeletal muscles into the subventricular zone of adult rats.

Stem cells isolated from adult tissues may be useful for autologous cell therapy in the nervous system. In the present study we tested the ability of multipotent stem cells isolated from adult muscle to survive and respond to migratory and differentiating cues when transplanted into the adult subventricular zone (SVZ). Prior to transplantation the cells were grown as spheres that expressed doublecortin, nestin, and betaIII-tubulin, as well as the mRNAs for the receptor EphA4 and the ligands ephrin B1, ephrin B2, but not ephrin B3. Four weeks after transplantation into the anterior part of the SVZ in adult rats, surviving cells were observed along the ventricular wall, in the SVZ, and in the posterior rostral migratory stream (RMS). None of these cells stained for betaIII-tubulin or doublecortin, which are molecules expressed by migrating neuroblasts, and none were present in the more rostral regions of the RMS or the olfactory bulb. However, most surviving transplanted cells were integrated into the wall of the lateral ventricle and expressed vimentin, a marker also expressed by ependymocytes. No tumors were observed 4 weeks posttransplantation. Our results suggest that multipotent stem cells isolated from adult muscle, which can be easily and safely isolated from patients and rapidly expanded ex vivo, may provide autologous vectors for the local delivery of secreted factors to the ventricles or nearby regions.

A phase 1 clinical trial of nerve growth factor gene therapy for Alzheimer disease.

Cholinergic neuron loss is a cardinal feature of Alzheimer disease. Nerve growth factor (NGF) stimulates cholinergic function, improves memory and prevents cholinergic degeneration in animal models of injury, amyloid overexpression and aging. We performed a phase 1 trial of ex vivo NGF gene delivery in eight individuals with mild Alzheimer disease, implanting autologous fibroblasts genetically modified to express human NGF into the forebrain. After mean follow-up of 22 months in six subjects, no long-term adverse effects of NGF occurred. Evaluation of the Mini-Mental Status Examination and Alzheimer Disease Assessment Scale-Cognitive subcomponent suggested improvement in the rate of cognitive decline. Serial PET scans showed significant (P < 0.05) increases in cortical 18-fluorodeoxyglucose after treatment. Brain autopsy from one subject suggested robust growth responses to NGF. Additional clinical trials of NGF for Alzheimer disease are warranted.

Fate of cloned embryonic neuroectodermal cells implanted into the adult, newborn and embryonic forebrain.

NE-4C, one-cell derived neuroectodermal stem cells expressing a reporter gene--green fluorescent protein (GFP) or heat-resistant alkaline phosphatase (PLAP)--or prelabeled with bromodeoxyuridine (BrdU) were implanted into the forebrain of adult, new-born and fetal mice and into the mid- and forebrain vesicles of early chick embryos. The fate of implanted cells in the mouse and chick hosts was followed up to 6 and 2 weeks, respectively. Neural differentiation was monitored by detecting the expression of neuron-specific markers and GFAP. NE-4C cells integrated into the early embryonic brain tissue and developed into morphologically differentiated neurons. The same cells produced expanding tumor-like aggregates in the newborn forebrain and were expelled from the adult forebrain parenchyma. In the adult brain, long-term survival and integration of stem cells were revealed only in neurogenic zones. The data suggest that noncommitted, proliferating neuroectodermal progenitors can integrate into the brain tissue at time and site of tissue genesis.

Human skin-derived stem cells migrate throughout forebrain and differentiate into astrocytes after injection into adult mouse brain.

Recent evidence indicates that neural stem cell properties can be found among a mammalian skin-derived multipotent population. A major barrier in the further characterization of the human skin-derived neural progenitors is the inability to isolate this population based on expression of cell surface markers. Our work has been devoted to purified human skin-derived stem cells that are capable of neural differentiation, based on the presence or absence of the AC133 cell surface marker. The enriched skin-derived AC133(+) cells express the CD34 and Thy-1 antigens. These cells cultured in a growth medium containing epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) proliferate, forming spheres, and differentiate in vitro into neurons, astrocytes, and rarely into oligodendrocytes. Single cells from sphere cultures initiated from human purified AC133(+) cells were replated as single cells and were able to generate new spheres, demonstrating the self-renewing ability of these stem cell populations. Brain engraftment of cells obtained from human purified AC133(+)-derived spheres generated different neural phenotypes: immature neurons and a most abundant population of well differentiated astrocytes. The AC133-derived astrocytes assumed perivascular locations in the frontal cortex. No donor-derived oligodendrocytes were found in the transplanted mouse brains. Several donor small, rounded cells that expressed endothelial markers were found close to the host vessel and near the subventricular zone. Thus, mammalian skin AC133-derived cells behave as a multipotent population with the capacity to differentiate into neural lineages in vitro and, prevalently, endothelium and astrocytes in vivo, demonstrating the great plasticity of these cells and suggesting potential clinical application.

Highly reproducible rat model of reversible forebrain ischemia--modified four-vessel occlusion model and its metabolic feature.

BACKGROUND: The four-vessel occlusion method introduced by Pulsinelli et al. is widely used as an experimental model for reversible forebrain ischemia in rats. METHOD: In this study, we further developed highly reproducible model of reversible forebrain ischemia. Under the microscope the visible vertebral arteries at the second vertebra could be easily electrocauterized and completely cut to yield complete cessation of circulation of both vertebral arteries. After 24 hours, male Wistar rats were subjected to 15, 30 and 45 minutes of forebrain ischemia by occluding both common carotid arteries with Sugita's temporary clips. (31)P-magnetic resonance spectra ((31)P-MRS) and (1)H-magnetic resonance images ((1)H-MRI) were obtained with a 6.3-T spectrometer to investigate sequential change of the in vivo brain metabolism. Electroencephalogram and the cortical blood flow by laser Doppler flowmetry were measured during ischemia and recirculation. Determination of endogenous superoxide scavenging activity in the brain cortex was performed by electron spin resonance spectrometry. FINDINGS: Brain water contents evaluated by the dry-wet weight method were increased at 1 hour and 48 hours after recirculation, which were demonstrated by (1)H-MRI. The superoxide scavenging activity showed a significant decrease at 45 minutes of recirculation and a significant increase at 12 hours of recirculation. The present modified model demonstrated that the mortality rates by 72 hours were 8.3% (15 minutes ischemia), 15.0% (30 minutes ischemia), and 42.9% (45 minutes ischemia), all of which were higher than that of the original method described by Pulsinelli et al. INTERPRETATION: In conclusion, this modified four-vessel occlusion method gives a high level of success in producing reversible forebrain ischemia.