Effect of hypoxia on the retina and superior colliculus of neonatal pigs.

PURPOSE: To evaluate the effect of hypoxia on the neonatal pig retina and brain, we analysed the retinal ganglion cells (RGCs) and neurons in the superior colliculus, as well as the response of astrocytes in both these central nervous system (CNS) structures. METHODS: Newborn pigs were exposed to 120 minutes of hypoxia, induced by decreasing the inspiratory oxygen fraction (FiO2: 10-15%), followed by a reoxygenation period of 240 minutes (FiO2: 21-35%). RGCs were quantified using Brn3a, a specific nuclear marker for these cells, and apoptosis was assessed through the appearance of active caspase-3. A morphometric analysis of the cytoskeleton of astrocytes (identified with GFAP) was performed in both the retina and superior colliculus. RESULTS: Hypoxia produced no significant change in the RGCs, although, it did induce a 37.63% increase in the number of active caspase-3 positive cells in the superior colliculus. This increase was particularly evident in the superficial layers of the superior colliculus, where 56.93% of the cells were positive for active caspase-3. In addition, hypoxia induced changes in the morphology of the astrocytes in the superior colliculus but not in the retina. CONCLUSIONS: Hypoxia in the neonatal pig does not affect the retina but it does affect more central structures in the brain, increasing the number of apoptotic cells in the superior colliculus and inducing changes in astrocyte morphology. This distinct sensibility to hypoxia may pave the way to design specific approaches to combat the effects of hypoxia in specific areas of the CNS.

Blindsight mediated by an S-cone-independent collicular pathway: an fMRI study in hemispherectomized subjects.

The purpose of our study was to investigate the ability to process achromatic and short-wavelength-sensitive cone (S-cone)-isolating (blue-yellow) stimuli in the blind visual field of hemispherectomized subjects and to demonstrate that blindsight is mediated by a collicular pathway that is independent of S-cone inputs. Blindsight has been described as the ability to respond to visual stimuli in the blind visual field without conscious awareness [Weiskrantz, L., Warrington, E. K., Sanders, M. D., & Marshall, J. Visual capacity in the hemianopic field following a restricted occipital ablation. Brain, 97, 709-728, 1974]. The roles of the subcortical neural structures in blindsight, such as the pulvinar and the superior colliculus, have been debated and an underlying neural correlate has yet to be confirmed. Using fMRI, we tested the ability to process visual stimuli that isolated the achromatic and short-wavelength-sensitive (S-)-cone pathways in three subjects: one control subject, one hemispherectomized subject with blindsight, and one hemispherectomized subject without blindsight. We demonstrated that (1) achromatic and S-cone-isolating stimuli presented to the normal visual hemifield of hemispherectomized subjects and to both visual hemifields of the control subject activated contralateral visual areas (V1/V2), as expected; (2) achromatic stimulus presentation but not S-cone-isolating stimulus presentation to the blind hemifield of the subject with blindsight activated visual areas FEF/V5; (3) whereas the cortical activation of the control subject was enhanced by an additional stimulus (achromatic and S-cone isolating) presented in the contralateral visual field, activation pattern of the subject with blindsight was enhanced by achromatic stimuli only. We conclude that the human superior colliculus is blind to the S-cone-isolating stimuli, and blindsight is mediated by an S-cone-independent collicular pathway.

Combined functional MRI and diffusion tensor imaging analysis of visual motion pathways.

BACKGROUND: Motion perception may be preserved after damage to striate cortex (primary visual cortex, area V1). Awareness and normal discrimination of fast-moving stimuli have been observed even in the complete absence of V1. These facts suggest that motion-sensitive cortex (the V5/MT complex or V5/MT+) may be activated by direct thalamic or collicular inputs that bypass V1. Such projections have been identified previously in monkeys but have not been shown in humans using neuroimaging techniques. METHODS: We used diffusion tensor imaging (DTI) tractography to visualize white matter fiber tracts connecting with V5/MT+ in 10 healthy volunteers. V5/MT+ was localized for each subject using functional MRI (fMRI). Functional activity maps were overlaid on high-resolution anatomical images and registered with the diffusion-weighted images to define V5/MT+ as the region of interest (ROI) for DTI tractography analysis. Fibers connecting to V1 were excluded from the analysis. RESULTS: Using conservative tractography parameters, we found connections between the V5/MT+ region and the posterior thalamus and/or superior colliculus in 4 of 10 subjects. CONCLUSIONS: Connections between the V5/MT+ region and the posterior thalamus and/or superior colliculus may explain visual motion awareness in the absence of a functioning V1.

Activation of superior colliculi in humans during visual exploration.

BACKGROUND: Visual, oculomotor, and - recently - cognitive functions of the superior colliculi (SC) have been documented in detail in non-human primates in the past. Evidence for corresponding functions of the SC in humans is still rare. We examined activity changes in the human tectum and the lateral geniculate nuclei (LGN) in a visual search task using functional magnetic resonance imaging (fMRI) and anatomically defined regions of interest (ROI). Healthy subjects conducted a free visual search task and two voluntary eye movement tasks with and without irrelevant visual distracters. Blood oxygen level dependent (BOLD) signals in the SC were compared to activity in the inferior colliculi (IC) and LGN. RESULTS: Neural activity increased during free exploration only in the SC in comparison to both control tasks. Saccade frequency did not exert a significant effect on BOLD signal changes. No corresponding differences between experimental tasks were found in the IC or the LGN. However, while the IC revealed no signal increase from the baseline, BOLD signal changes at the LGN were consistently positive in all experimental conditions. CONCLUSION: Our data demonstrate the involvement of the SC in a visual search task. In contrast to the results of previous studies, signal changes could not be seen to be driven by either visual stimulation or oculomotor control on their own. Further, we can exclude the influence of any nearby neural structures (e.g. pulvinar, tegmentum) or of typical artefacts at the brainstem on the observed signal changes at the SC. Corresponding to findings in non-human primates, our data support a dependency of SC activity on functions beyond oculomotor control and visual processing.

Analysis of microvascular trees by means of scanning electron microscopy of vascular casts and 3D-morphometry.

Arterial and capillary trees form by consecutive branching (mostly bifurcations) from a stem vessel, venous trees form by repeated merging of blood vessels. Diameters of stem (parent, mother) vessels and daughter vessels (branches), interbranching distances and branching angles between stem and daughter vessels lastly define the overall three-dimensional structure of the vascular network as well as the basic transport capacity of the system. Here we use scanning electron microscopy and 3D-morphometry to measure these variables from stereo paired images of vascular corrosion casts of the anterior cerebral artery and its main branches and from arteriolar bifurcations of the mesencephalic optic tectum in the actinopterygian fish, Acipenser ruthenus. We then calculate bifurcation indices, area ratios, asymmetry ratios and test for the optimality principles underlying the bifurcations studied. Our results show that arteriolar bifurcations in the optic tectum are in favor of the principles of minimum pumping power and minimum volume rather than the principles of minimum surface and minimum drag. We conclude that scanning electron microscopy of vascular corrosion casts in conjunction with 3D-morphometry is an excellent tool to thoroughly analyze vascular trees in healthy and diseased tissues and organs, as well as on an ontogenetic and phylogenetic scale.

Visual responses of the human superior colliculus: a high-resolution functional magnetic resonance imaging study.

The superior colliculus (SC) is a multimodal laminar structure located on the roof of the brain stem. The SC is a key structure in a distributed network of areas that mediate saccadic eye movements and shifts of attention across the visual field and has been extensively studied in nonhuman primates. In humans, it has proven difficult to study the SC with functional MRI (fMRI) because of its small size, deep location, and proximity to pulsating vascular structures. Here, we performed a series of high-resolution fMRI studies at 3 T to investigate basic visual response properties of the SC. The retinotopic organization of the SC was determined using the traveling wave method with flickering checkerboard stimuli presented at different polar angles and eccentricities. SC activations were confined to stimulation of the contralateral hemifield. Although a detailed retinotopic map was not observed, across subjects, the upper and lower visual fields were represented medially and laterally, respectively. Responses were dominantly evoked by stimuli presented along the horizontal meridian of the visual field. We also measured the sensitivity of the SC to luminance contrast, which has not been previously reported in primates. SC responses were nearly saturated by low contrast stimuli and showed only small response modulation with higher contrast stimuli, indicating high sensitivity to stimulus contrast. Responsiveness to stimulus motion in the SC was shown by robust activations evoked by moving versus static dot stimuli that could not be attributed to eye movements. The responses to contrast and motion stimuli were compared with those in the human lateral geniculate nucleus. Our results provide first insights into basic visual responses of the human SC and show the feasibility of studying subcortical structures using high-resolution fMRI.

Microsurgical anatomy of the venous drainage of the mesencephalodiencephalic junction.

OBJECTIVE: The veins draining the posterior wall of the third ventricle and its adjacent structures and the posterior part of the midbrain have been the most neglected of the intracranial vascular structures in both the anatomic and neurosurgical literature. During our dissections of the pineal region and the quadrigeminal cistern, we did not always encounter topographic anatomy as described in previous articles. The purpose of this study is to describe the topographic anatomy and normal variations of the specific veins that drain the collicular plate and the pineal body and their adjacent structures with a view to better defining neurosurgical approaches to the pineal region. METHODS: The deep cerebral veins draining the pineal body, the collicular plate, and their surrounding neural structures were examined on both sides of 25 adult cadaveric brains. In all specimens, the carotid and vertebral arteries and the jugular veins were perfused with red or blue silicone, respectively, to facilitate dissection under x3 to x40 magnification. RESULTS: The venous plexus on the dorsal aspect of the collicular plate drains via collicular veins according to three different patterns. These types of drainage are closely related to the existence or absence of the basal vein on one or both sides. CONCLUSION: The veins draining the superior and inferior aspects of the pineal body form a superior and an inferior pineal vein that usually drain into the internal or great cerebral vein.

Brain phagocytes may empty tissue debris into capillaries.

The possibility that brain phagocytes may empty remnants of degenerated neurons into capillaries has been studied in frogs. Degeneration of nerve fibers was brought about by transectioning the optic tract, the tectothalamic and tectoisthmic tracts, the postoptic commissure or the radial nerve. To help identification of phagocytozed degenerated neuronal elements, the transected fibers were filled either with horseradish peroxidase (HRP) or cobaltous-lysine complex. The survival times were 3, 4, 7, 27, 47 and 70 days after the application of the markers. The HRP-labeled structures were identified in 60 microm thick sections using diaminobenzidine as chromogen, while cobalt was precipitated in the form of cobaltous sulfide. Small pieces of these sections were further processed for electron microscopy. In each area of the brain and spinal cord investigated, microglial cells and astrocytic processes containing fragments of degenerated neuronal elements could be seen close to capillaries. In some cases a microglial or astrocytic process pierced the capillary basal lamina and seemingly delivered inclusion bodies into the cytoplasm of capillary endothelial cells and pericytes. In the inclusion bodies, which were usually large vesicles, fragments of HRP or cobalt-labeled or unlabeled membranes with a foamy appearance, or condensed myelin lamellae could be observed. These vesicles protruded the luminal membrane of the endothelial cell that was disrupted in some cases suggesting that the content of the inclusion body was discharged into the lumen of the capillary. These results give support to Penfield's hypothesis (1925) that glial cells may empty phagocytozed materials into capillaries.

Functional human heme oxygenase has a neuroprotective effect on adult rat ganglion cells after pressure-induced ischemia.

Adult retina subjected to transient ischemia and reperfusion leads to controlled retinal ganglion cell (RGC) death over a period. Modification of intracellular mechanisms through a specific adenoviral vector containing the hemoxygenase gene (HO-1) provides avenues for RGC survival following HO-1 gene transfer and ischemia. RGC death rate was reduced by an average of 15% at 1, 2 and 3 weeks. A significant number of RGC transfected with functional HO-1 survived ischemic insults. Pharmacological stimulation of HO-1 may constitute a novel therapeutic approach to rescuing RGC experiencing ischemic/reperfusion injury.

Functional neuroanatomy of smooth pursuit and predictive saccades.

We used PET to study differences in cerebral blood flow (CBF) in smooth pursuit, predictive saccades and fixation. Eye movements were monitored in the scanner. Compared with fixation, pursuit and predictive saccades activated a network of highly similar areas, including frontal eye fields, supplementary eye fields, V5 and medial cuneus. Our findings are consistent with non-human primate studies that suggest that pursuit and saccades are controlled by similar and adjacent neural areas. Pursuit was associated with greater activation of caudate than saccades, suggesting a role for basal ganglia in pursuit that is consistent with studies of neurological populations. Saccades were associated with greater activation of cerebellum and frontal eye fields. A frontal-cerebellar loop may be important in coordinating the preparation and timing of saccades in predictive tracking.

Brain stem blood flow, pupillary response, and outcome in patients with severe head injuries.

OBJECTIVE: Acute pupillary dilation in a head-injured patient is a neurological emergency. Pupil dilation is thought to be the result of uncal herniation causing mechanical compression of the IIIrd cranial nerve and subsequent brain stem compromise. However, not all patients with herniation have fixed and dilated pupils, and not all patients with nonreactive, enlarged pupils have uncal herniation. Therefore, we have tested an alternative hypothesis that a decrease in brain stem blood flow (BBF) is a more frequent cause of mydriasis and brain stem symptomatology after severe head injury. We determined the relation of BBF to outcome and pupillary response in patients with severe head injuries. METHODS: One hundred sixty-two patients with a Glasgow Coma Scale score of 8 or less underwent stable xenon computed tomographic blood flow determination at the level of the superior colliculus, and this blood flow was correlated with pupillary features, intracranial pressure, computed tomographic scan pathology, and outcome. RESULTS: A BBF of less than 40 ml/100 g/min was significantly associated with poor outcome (P < 0.009). In patients with bilaterally nonreactive pupils, the BBF was 30.5+/-16.8 ml/100 g/min, and in those with normally reactive pupils, the BBF was 43.8+/-18.7 ml/100 g/min (P < 0.001). Intracranial pressure and the presence of a brain stem lesion observed on the computed tomographic scan did not correlate with BBF, pupillary size, or reactivity. Unfavorable outcome at 12 months was directly related to age (P = 0.062) and inversely related to pupillary responsiveness (P = 0.0006), pupil size (P = 0.005), and BBF of less than 40 ml/100 g/min (P = 0.009). CONCLUSION: These findings suggest that pupillary dilation is associated with decreased BBF and that ischemia, rather than mechanical compression of the IIIrd cranial nerve, is an important causal factor. More important, pupil dilation may be an indicator of ischemia of the brain stem. If cerebral blood flow and cerebral perfusion pressure can be rapidly restored in the patient with severe head injury who has dilated pupils, the prognosis may be good.

Ultrastructural localization of lectin binding sites in the developing brain microvasculature.

The temporo-spatial patterning of lectin-binding sites was examined by lectin histochemistry and quantitative methods in the microvasculature of the optic tectum of 9-, 14-, 20-day-old embryos and 30-day-old chickens. Horseradish peroxidase and colloidal-gold-labelled lectins were used for detection of beta-D-galactose (RCA-I, Ricinus communis agglutinin-I) and of N-acetylglucosamine and sialic residues (WGA, Wheat germ agglutinin) at light and electron microscopical levels. At the light microscopical level, RCA-I and WGA binding sites were detectable in the early embryonic capillaries in a diffuse staining pattern; in later embryonic stages and in adult animals, RCA-I labelling became located on the abluminal surface of the vessels, while WGA staining was detected on the luminal surface. Ultrastructurally, gold labelling for RCA-I was seen intracytoplasmically in endothelial cells in 9-day-old embryos. In 14-to 20-day-old embryos and in chickens, binding sites for RCA-I were detected in endothelial tight junctions and basement membranes. In contrast, labelling of the gold-coupled WGA lectin was distributed almost exclusively on the luminal endothelial surface already in early embryos. The results indicate that the endothelial cells of the optic tectum acquire functional polarity early in their development and that glycoconjugates containing beta-D-galactose residues are involved in the biochemical composition of the tight junctions and basement membrane, which are considered to be key structures in blood-brain barrier (BBB) differentiation.

Cavernous angioma with olivary hypertrophy and contralateral cerebellar diaschisis.

We describe a 66-year-old man with a 20-year history of ataxic gait who suddenly developed diplopia on rightward gaze. Neurologic examination revealed right hemi-ataxia and hemi-hypesthesia, and left internuclear ophthalmoplegia. MRI showed a cavernous angioma in the left tectum, mild right cerebellar atrophy, and left interior olivary hypertrophy. Single photon emission computed tomography (SPECT) imaging demonstrated contralateral cerebellar diaschisis. We discuss the findings and review the literature concerning contralateral cerebellar diaschisis.

Morphological aspects of the vascularization in intraventricular neural transplants from embryo to embryo.

Intraventricular transplants of neural tissues were performed in ovo from embryo to embryo. Fragments of the nervous wall of the optic lobe (tectum) from 14-day chick or 12-day quail embryos (donor) were inserted into the ventricle of the right optic lobe of 6-day chick or 5-day quail embryos (host). Chick-to-chick, chick-to-quail and quail-to-chick grafts were carried out. The vascularization changes occurring in the host tectum and in the grafted neural tissues were analysed under light, transmission, and scanning electron microscopes and by morphometric methods. In the host embryo tectum, the neural graft stimulates a statistically significant increment in vessel density and a vessel sprouting into the ventricle of the optic lobe. The vascular sprouts reach the transplanted tissue and establish connections with its native microvasculature. The chick-to-quail and quail-to-chick grafts, submitted to immunoreaction with a quail-specific antibody which recognizes an antigen (MB1) present on endothelial cells, indicate that re-establishment of the circulation in the graft depends upon anastomoses between host and donor vasculatures and the rapid new growth of host-derived and donor-native vessels. The presence of macrophage-like cells escorting the new-growing vessels suggests that these cells are involved in the host and donor tissue angiogenesis.

A compared TEM/SEM investigation on the pericytic investment in developing microvasculature of the chick optic tectum.

The pericytes have been ultrastructurally and morphometrically analyzed in the neural vessels of the chick embryo optic tectum, under the transmission and scanning electron microscopes. The observations demonstrated that shape and surface features of pericytes, as well as their spatial relation with the endothelium, remarkably change during development, whereas their ultrastructure does not substantially modify from the early to the late embryonic stages. The pericytes have an ovoid body, broad processes, smooth surfaces, and are closely applied to the endothelial tube on days 5-7; they show convolute shape, highly irregular surfaces, and are complicately interdigitated with the endothelial cells, when a vivacious vessel growth takes place, on days 12-14; finally, they are flattened, smooth, highly branched, and completely enclosed in the basement lamina on days 20-21, when a definitive vascular pattern is established. The contribution of pericytes to the formation of the basement lamina has been confirmed by the detection of exocytotic vesicles discharging their content toward the subendothelial cleft. The morphometric evaluations revealed that pericytes provide the growing neural microvessels with an almost continuous coverage which, however, undergoes a significant reduction within hatching time. On the whole, the results suggest that the pericytes are as active as the endothelial cells during the vessel growth and play an inhibitory role on the endothelial proliferation only later on, when they are closely adherent to the endothelium and are encompassed by the basement lamina.

Immunolocalization of exoglycoproteins ("ependymins") in the goldfish brain.

Exoglycoproteins (X-GPs) are a group of very abundant soluble glycoproteins in the goldfish brain. Immunostaining with polyclonal antisera to X-GPs revealed consistent perinuclear staining in the cells of the inner and intermediate layers of the leptomeninx, which is homologous to the pia-arachnoid. Immunolabelling was also prominent in the outer wall of capillaries, and in a variable population of 10-12 microns granular cells that appeared mainly near the ventricles and occasionally within the ventricles or under the meninges. In some cases, small and medium-sized lymphocytes were immunostained. Lymphocytes were sometimes associated with the granular cells, which may be hematogenous cells in transit toward the ventricles. The choroid plexus, saccus dorsalis, the roof of the third ventricle and Reissner's fiber showed strong immunostaining. The localization of the X-GPs suggests that they may contribute to maintenance of the blood-brain barrier or to regulation of immune function within the brain.

Transient increase in the in vivo binding of the benzodiazepine antagonist [3H]flumazenil in deafferented visual areas of the adult mouse brain.

Flumazenil is an imidazobenzodiazepine, an antagonist of central benzodiazepine (BDZ) receptors. BDZ binding sites are a modulatory component located on the gamma-aminobutyric acid (GABA) receptor macromolecule. We studied the effect of monocular enucleation on [3H]flumazenil binding in deprived and intact visual areas and nonvisual areas of the adult mouse brain under in vivo conditions. [3H]flumazenil binding was examined at seven time points up to 56 days postenucleation. In some monocularly deprived mice, changes in local blood flow accompanied with the BDZ receptor response were evaluated by coinjection of [3H]flumazenil and 99mTc-HMPAO. Monocular enucleation produced a transient increase in [3H]flumazenil binding in the deprived visual cortex and superior colliculus. At 17 days postenucleation, [3H]flumazenil binding in the anterior and posterior portions of the visual cortex and the superior colliculus increased by 28%, 15% and 23%, respectively, and declined to control levels at 45 days postenucleation. The increase in [3H]flumazenil was accompanied with a decrease in blood flow. Alterations in BDZ receptors and blood flow were selective to deprived visual structures. The regional correlation between the metabolic deficit and the BDZ response provides further support that the increase in BDZ receptor binding is confined to regions of reduced neuronal activity. [11C]flumazenil is an excellent radiotracer for in vivo imaging of benzodiazepine receptors in human brain using positron emission tomography (PET). This study suggests the suitability of [11C]flumazenil for in vivo PET study of BDZ receptor response to deafferentation of visual structures in human brain.

An adenosine agonist increases blood flow and density of capillary branches in the optic tectum of Xenopus laevis tadpoles.

OBJECTIVE: To test the hypothesis that adenosine can increase capillary densities in developing brain tissue. METHODS: Transparent tadpoles of albino Xenopus laevis were exposed to adenosine agonists, mainly 5'-N-ethylcarboxamidoadenosine (NECA), and to antagonists, mainly 8-phenyltheophylline (8PT) in aquarium water. After 2 weeks in drugs, networks of blood vessels on the dorsal surface of the optic tectum were scanned in vivo by videomicroscopy. Densities of surface capillaries and venules, of deep branches, and of deep perfused capillaries were calculated. RESULTS: NECA initially dilated brain blood vessels and chronically increased blood flow by a simple subjective index. 8PT diminished diameters and prevented the subjective flow increase. Chronic 3 microM NECA significantly increased densities of total deep branches from pial vessels into the tectum. CONCLUSIONS: In an in vivo amphibian assay, NECA dilated brain capillaries and venules and increased their flow and density. Adenosine was present chemically and increased during metabolic stress. These results are consistent with adenosine as a metabolic signal for growth of blood vessels during brain development. In addition, it appeared that short-term dilation and flow increase in tectal capillaries in acute NECA was followed over a period of weeks in chronic NECA by vascular remodeling and return of diameters to normal.

Vimentin- and GFAP-immunoreactivity in developing and mature neural microvessels. Study in the chicken tectum and cerebellum.

The expression of the cytoskeletal filaments vimentin and GFAP has been analyzed by immunocytochemical techniques in endothelial cells, pericytes, and astrocyte perivascular endfeet of microvessels of chicken optic tectum and cerebellum during embryonic development and in adulthood. Endothelial cells and pericytes were characterized by strong vimentin-immunoreactivity in both tectum and cerebellum only in early developmental stages (11-15 incubation days, i.d.). Astrocyte processes closely associated with the vessel wall were vimentin stained in the 11 i.d. cerebellum and vimentin-and GFAP-reactive in 15 i.d. tectum. These perivascular endfeet became GFAP-immuno-stained in the tectum and cerebellum by the 21st i.d. The results indicate that intermediate filament expression in the cells of the brain microvasculature is developmentally regulated, and suggest that the vimentin to GFAP transition in perivascular astrocytes parallels the vessel wall maturation.