Jens Christian Sørensen

Memory impaired aged rats: No loss of principal hippocampal and subicular neurons

A group of 52 male, 2-year-old Ico:WIST rats were tested on the spatial reference memory version of the Morris water maze. Their performance was rated by comparisons with the performance of 2.5-month-old control rats. Unbiased stereological estimates were made of the number of neurons in the major subdivisions of the hippocampus proper and the subiculum of the 5 aged rats with the most impaired performance, the 5 aged rats with the best performance, and 5 young control rats. There were no significant differences between the mean numbers of neurons in the various subdivisions of the hippocampal region of the impaired and nonimpaired aged groups and similarly no decreases in neuron numbers in the pooled group of aged rats relative to the control rats. The results indicate that, in rats, the structural correlates of age-related deficits in spatial memory are to be found in parameters other than the number of neurons in the hippocampus proper and the subiculum.

Influence of an enriched environment and cortical grafting on functional outcome in brain infarcts of adult rats

The purpose of this work was to study if enriched housing conditions and fetal neocortical transplantation could enhance the functional outcome after focal brain ischemia in adult rats. The right middle cerebral artery (MCA) was ligated in 34 inbred, spontaneously hypertensive male rats, which were then randomly divided into three groups. Groups A and B were transferred to an enriched environment, i.e., a large cage with opportunities for various activities but not forcing the rats to do any particular tasks; group C was kept in standard laboratory cages. Three weeks after the MCA occlusion blocks of fetal neocortical tissue (Embryonic Day 17) were transplanted to the infarct cavity in groups B and C. Rats in group A (n = 11) and group B (n = 11) performed equally well and significantly better than rats in group C (n = 10) when placed on an inclined plane and when traversing a rotating pole 6 and 9 weeks after the MCA occlusion and in a leg placement test at 9, but not 6 and 12 weeks. Skilled forelimb function did not differ between the groups. Infarct size and thalamic atrophy did not differ between the groups and graft size was similar in group B and C. There was no correlation between infarct size and motor function in any of the tests in rats housed in an enriched environment. Since the environment can significantly alter functional outcome without reducing infarct size we suggest that more attention should be given to the role of the laboratory environment and to long term behavioral outcome in experimental stroke.

The role of the microcirculation in delayed cerebral ischemia and chronic degenerative changes after subarachnoid hemorrhage.

The mortality after aneurysmal subarachnoid hemorrhage (SAH) is 50%, and most survivors suffer severe functional and cognitive deficits. Half of SAH patients deteriorate 5 to 14 days after the initial bleeding, so-called delayed cerebral ischemia (DCI). Although often attributed to vasospasms, DCI may develop in the absence of angiographic vasospasms, and therapeutic reversal of angiographic vasospasms fails to improve patient outcome. The etiology of chronic neurodegenerative changes after SAH remains poorly understood. Brain oxygenation depends on both cerebral blood flow (CBF) and its microscopic distribution, the so-called capillary transit time heterogeneity (CTH). In theory, increased CTH can therefore lead to tissue hypoxia in the absence of severe CBF reductions, whereas reductions in CBF, paradoxically, improve brain oxygenation if CTH is critically elevated. We review potential sources of elevated CTH after SAH. Pericyte constrictions in relation to the initial ischemic episode and subsequent oxidative stress, nitric oxide depletion during the pericapillary clearance of oxyhemoglobin, vasogenic edema, leukocytosis, and astrocytic endfeet swelling are identified as potential sources of elevated CTH, and hence of metabolic derangement, after SAH. Irreversible changes in capillary morphology and function are predicted to contribute to long-term relative tissue hypoxia, inflammation, and neurodegeneration. We discuss diagnostic and therapeutic implications of these predictions.

Fetal Neocortical Tissue Blocks Implanted in Brain Infarcts of Adult Rats Interconnect with the Host Brain

The purpose of the present study was to study if the connectivity of fetal neocortical tissue blocks placed in ischemic brain infarcts of adult rats would be enhanced in rats housed in an enriched environment. We also investigated whether the enriched housing conditions could enhance the postischemic and postgrafting functional outcome, in terms of motor behavior. This part of the study has been published recently. The middle cerebral artery was ligated on the right side in 37 inbred, adult male spontaneously hypertensive rats. The rats were placed at random either in an enriched environment (groups A and B) or in standard laboratory cages (group C). Three weeks after the artery occlusion, blocks of fetal sensorimotor cortex (embryonic day 17) were transplanted into the infarct cavity of rats from groups B and C. After 9 weeks all transplanted rats received an injection, into the graft, of a mixture containing the two tracers Fluoro-Gold and biotinylated Dextran amine. The transplants revealed a structured morphology with whorls and bands of cells reminiscent of normal neocortex. Tracing of efferent transplant to host fibers with biotinylated Dextran amine showed pronounced intrinsic transplant projections, as well as fibers, although significantly fewer, to the host ipsilateral sensorimotor cortex, striatum, and thalamus. Host to transplant projections were revealed by Fluoro-Gold-labeled cells found in the ipsilateral host sensorimotor cortex, the basal nucleus of Meynert, the thalamic ventrobasal, ventrolateral and posterior nuclei, and in the dorsal raphe nuclei. We conclude that fetal frontal neocortical block grafts placed in brain infarcts of adult rats develop a morphology reminiscent of normal neocortex and that both afferent and efferent neural connections, although sparse, are established with the host brain, whether the rats are reared under enriched housing conditions or not.

Hippocampal pyramidal cells: the reemergence of cortical lamination

The increasing resolution of tract-tracing studies has led to the definition of segments along the transverse axis of the hippocampal pyramidal cell layer, which may represent functionally defined elements. This review will summarize evidence for a morphological and functional differentiation of pyramidal cells along the radial (deep to superficial) axis of the cell layer. In many species, deep and superficial sublayers can be identified histologically throughout large parts of the septotemporal extent of the hippocampus. Neurons in these sublayers are generated during different periods of development. During development, deep and superficial cells express genes (Sox5, SatB2) that also specify the phenotypes of superficial and deep cells in the neocortex. Deep and superficial cells differ neurochemically (e.g. calbindin and zinc) and in their adult gene expression patterns. These markers also distinguish sublayers in the septal hippocampus, where they are not readily apparent histologically in rat or mouse. Deep and superficial pyramidal cells differ in septal, striatal, and neocortical efferent connections. Distributions of deep and superficial pyramidal cell dendrites and studies in reeler or sparsely GFP-expressing mice indicate that this also applies to afferent pathways. Histological, neurochemical, and connective differences between deep and superficial neurons may correlate with (patho-) physiological phenomena specific to pyramidal cells at different radial locations. We feel that an appreciation of radial subdivisions in the pyramidal cell layer reminiscent of lamination in other cortical areas may be critical in the interpretation of studies of hippocampal anatomy and function.

Rapid disappearance of zinc positive terminals in focal brain ischemia

The study was undertaken to determine if the levels of vesicular zinc in neuronal terminals would decrease in response to focal brain ischemia. The middle cerebral artery was occluded distal to the striatal branches in male spontaneously hypertensive rats. At 7, 15, 30, 45, 60, 90, 120 min; 3, 6, 12, 24, 48 h and 7 days later the animals were sacrificed and the brains were stained for zinc-sulfides, cell bodies and AChE-positive cholinergic fibers. The density of zinc positive terminals significantly decreased in the neocortical ischemic zone 7 min after middle cerebral artery occlusion (MCAO). In the neocortical layers II and III most zinc positive neuronal terminals disappeared at 7 min after MCAO whereas the zinc positive terminals in layers V and VI remained positive at least 2 h. Beginning at 1 h after MCAO and progressing to 24 h a significant decrease in the density of zinc positive terminals was observed in the dorsolateral striatum, and ventrobasal thalamic nucleus, both major projection areas of the sensorimotor cortex. The disappearance of zinc positive neuronal terminals in the ischemic neocortex and related areas, is most likely due to a neuronal release of vesicular zinc in response to hypoxia. The high extracellular concentration of zinc is thought to be both neuroprotective by blocking the NMDA receptor and neurotoxic by activating neuronal influx of Ca2+ through voltage gated calcium channels. It seems evident that the latter effect of zinc is contributing to the neuronal death in focal brain ischemia.

Long-term Delivery of Nerve Growth Factor by Encapsulated Cell Biodelivery in the Göttingen Minipig Basal Forebrain

Nerve growth factor (NGF) prevents cholinergic degeneration in Alzheimers disease (AD) and improves memory in AD animal models. In humans, the safe delivery of therapeutic doses of NGF is challenging. For clinical use, we have therefore developed an encapsulated cell (EC) biodelivery device, capable of local delivery of NGF. The clinical device, named NsG0202, houses an NGF-secreting cell line (NGC-0295), which is derived from a human retinal pigment epithelial (RPE) cell line, stably genetically modified to secrete NGF. Bioactivity and correct processing of NGF was confirmed in vitro. NsG0202 devices were implanted in the basal forebrain of Göttingen minipigs and the function and retrievability were evaluated after 7 weeks, 6 and 12 months. All devices were implanted and retrieved without associated complications. They were physically intact and contained a high number of viable and NGF-producing NGC-0295 cells after explantation. Increased NGF levels were detected in tissue surrounding the devices. The implants were well tolerated as determined by histopathological brain tissue analysis, blood analysis, and general health status of the pigs. The NsG0202 device represents a promising approach for treating the cognitive decline in AD patients.

Distribution and morphology of serotonin-immunoreactive neurons in the brainstem of the New Zealand white rabbit

The aim of the present study was to demonstrate the morphology and distribution of the serotonergic neurons in the brainstem of the New Zealand white rabbit by using a highly specific immunocytochemical procedure. It was possible to divide the serotonergic neurons into a rostral group, which is situated in the mesencephalon and the rostral part of the pons containing four serotonergic nuclei, and a caudal group, which is located in the medulla and the caudal part of the pons containing five serotonergic nuclei. The localization of the serotonergic neurons is presented in a detailed brainstem atlas, and the distribution of the serotonergic neurons is in accordance with results obtained by other authors in different species.

Zinc-containing telencephalic connections to the rat striatum: a combined Fluoro-Gold tracing and histochemical study

The organization of telencephalic zinc-containing neurons projecting to the rat striatum was investigated by combining intrastriatal injections of the retrograde fluorescent tracer Fluoro-Gold with histochemistry revealing zinc-containing neurons and terminals. Throughout the ipsilateral and contralateral neocortex, corticostriatal zinc-containing neurons with striatal projections were located predominantly at the border between deep layer V and superficial layer VI. Additional, but fewer zinc-containing neurons were located in layers II, III and deep layer VI of the ipsilateral neocortex. The main neocortical source of zinc-containing afferents to the striatum were the frontal motor cortices. Smaller contingents of zinc-containing projections arose from the motor cortical forelimb and hindlimb areas and the parietal cortical areas. In the cingulate cortex, zinc-containing neurons with striatal projections were found predominantly in the ipsilateral layers II and III, with only few neurons in the ipsilateral layer VI and in the contralateral layers II, III and VI. Subcortically, zinc-containing neurons belonging to the amygdalostriatal projection were found bilaterally in the basolateral and basomedial nuclei of the amygdala. Zinc has been found to modulate the response of many ligand- and voltage-gated ion channels, including both GABA receptors and NMDA-, AMPA- and kainate-type glutamate receptors. The present findings raise the possibility that zinc in the corticostriatal projections might play a role in the selective, possibly excitotoxic, cell death of GABAergic projections seen in Huntingtons disease.

MRI-guided stereotaxic targeting in pigs based on a stereotaxic localizer box fitted with an isocentric frame and use of SurgiPlan computer-planning software

We present a stereotaxic procedure enabling MRI-guided isocentric stereotaxy in pigs. The procedure is based on the Leksell stereotaxic arch principle, and a stereotaxic localizer box with an incorporated fiducial marking system (sideplates) defining a stereotaxic space similar to the clinical Leksell system. The obtained MRIs can be imported for 3D-reconstruction and coordinate calculation in the clinical stereotaxic software planning system (Leksell SurgiPlan, Elekta AB, Sweden). After MRI the sideplates are replaced by a modified Leksell arch accommodating clinical standard manipulators for isocentric placement of DBS-electrodes, neural tracers and therapeutics in the calculated target coordinates. The mechanical accuracy of the device was within 0.3-0.5 mm. Stereotaxic MRIs were imported to the stereotaxic software planning system with a mean error of 0.4-0.5 mm and a max error of 0.8-0.9 mm. Application accuracy measured on a phantom and on inserted skull markers in nine pigs was within 1 mm in all planes. The intracerebral application accuracy found after placement of 10 manganese trajectories within the full extent of the intracerebral stereotaxic space in two minipigs was equally randomly distributed and within 0.7+/-0.4; 0.5+/-0.4; and 0.7+/-0.3mm in the X, Y, and Z plane. Injection of neural tracers in the subgenual gyrus of three minipigs and placement of encapsulated gene-modified cells in four minipigs confirmed the accuracy and functionality of the described procedure. We conclude that the devised technique and instrumentation enable high-precision stereotaxic procedures in pigs that may benefit future large animal neuroscience research and outline the technical considerations for a similar stereotaxic methodology in other animals.