The porcine corticospinal decussation: A combined neuronal tracing and tractography study.

BACKGROUND: Pigs and minipigs are increasingly used as non-primate large animal models for preclinical research on nervous system disorders resulting in motor dysfunction. Knowledge of the minipig pyramidal tract is therefore essential to support such models. AIM AND METHODS: This study used 5 female Göttingen minipigs aging 11-15 months. The Göttingen minipig corticospinal tract was investigated, in the same animals, with in vivo neuronal tracing and with postmortem diffusion weighted MRI tractography to provide a thorough insight in the encephalic distribution of this primary motor pathway and its decussation at the craniocervical junction. RESULTS: The two methods similarly outlined the course of the pyramidal tract from its origin in the motor cortex down through the internal capsule to the craniocervical junction, where both methods displayed an axonal crossover at the pyramid decussation. The degree of crossover was quantified with unbiased stereology, where 81-93% of the traced corticospinal fibers crossed to the contralateral spinal cord. Accordingly, in the upper cervical spinal cord the corticospinal tract is primarily distributed in the contralateral lateral funiculus and in close relation to the gray matter, wherein some direct terminations on large ventral column gray matter neurons could be identified. DISCUSSION: The combination of neuronal tracing and tractography exploited the strengths of the respective methods to gain a better understanding of the encephalic distribution and craniocervical decussation of the Göttingen minipig corticospinal tract. Moreover, a quantification of the crossing fibers was obtained from the tracing data, which was not possible with tractography. Our data indicate that the porcine corticospinal system is quite lateralized down to the investigated upper cervical levels. However, further elucidation of this point will require a full examination of the corticospinal tracing pattern into the caudal spinal cord combined with an analysis of the direct versus indirect termination pattern on the lower motor neurons.

Space Balls Revisited: Stereological Estimates of Length With Virtual Isotropic Surface Probes.

The space ball probe was fully described in the literature 15 years ago by Mouton et al. (2002). Since then, it has been used in a number of studies in the nervous system that focus on axon, dendrite, and capillary length. The length of structural parameters in tissues reflect functional aspects of the tissues. Here, some of the various applications of this methodology will be presented, along with a review of the salient features of the methodology that has resulted in new wave of quantitative morphological studies of length in the nervous system. The validity of the method is discussed in view of its widespread use along with insights into the problems associated with its application to histological tissue and future techniques for applying space balls.

Disturbances in the control of capillary flow in an aged APPswe/PS1ΔE9 model of Alzheimer's disease.

Vascular changes are thought to contribute to the development of Alzheimer's disease, and both cerebral blood flow and its responses during neural activation are reduced before Alzheimer's disease symptoms onset. One hypothetical explanation is that capillary dysfunction reduces oxygen extraction efficacy. This study compares the morphology and hemodynamics of the microvasculature in the somatosensory cortex of 18-month-old APPSWE/PS1ΔE9 (transgenic [Tg]) mice and wild-type (WT) littermates. In particular, the extent to which their capillary transit times homogenize during functional activation was measured and compared. Capillary length density was similar in both groups but capillary blood flow during rest was lower in the Tg mice, indicating that cortical oxygen availability is reduced. The capillary hemodynamic response to functional activation was larger, and lasted longer in Tg mice than in WT mice. The homogenization of capillary transit times during functional activation, which we previously demonstrated in young mice, was absent in the Tg mice. This study demonstrates that both neurovascular coupling and capillary function are profoundly disturbed in aged Tg and WT mice.

Neuron and neuroblast numbers and cytogenesis in the dentate gyrus of aged APPswe/PS1dE9 transgenic mice: Effect of long-term treatment with paroxetine.

Altered neurogenesis may influence hippocampal functions such as learning and memory in Alzheimer's disease. Selective serotonin reuptake inhibitors enhance neurogenesis and have been reported to reduce cerebral amyloidosis in both humans and transgenic mice. We have used stereology to assess the longitudinal changes in the number of doublecortin-expressing neuroblasts and number of granular neurons in the dentate gyrus of APPswe/PS1dE9 transgenic mice. Furthermore, we investigated the effect of long-term paroxetine treatment on the number of neuroblasts and granular neurons, hippocampal amyloidosis, and spontaneous alternation behaviour, a measure of spatial working memory, in transgenic mice. We observed no difference in granular neurons between transgenic and wild type mice up till 18months of age, and no differences with age in wild type mice. The number of neuroblasts and the performance in the spontaneous alternation task was reduced in aged transgenic mice. Paroxetine treatment from 9 to 18months of age reduced hippocampal amyloidosis without affecting the number of neuroblasts or granular neurons. These findings suggest that the amyloidosis affects the differentiation of neuroblasts and spatial working memory, independent of changes in total granular neurons. Furthermore, while long-term paroxetine treatment may be able to reduce hippocampal amyloidosis, it appears to have no effect on total number of granular neurons or spatial working memory.

Stereological investigation of the CA1 pyramidal cell layer in untreated and lithium-treated 3xTg-AD and wild-type mice.

Pyramidal neuron loss in the hippocampal CA1 region is a very early hallmark of Alzheimer disease (AD). Lithium might be a therapeutic strategy for AD due to its neuroprotective and neurotrophic properties. This study used modern stereological techniques to investigate possible CA1 pyramidal neuron loss in 11-month-old triple transgenic AD (3xTg-AD) mice, and also the effects of therapeutic and subtherapeutic lithium doses on the number and density of CA1 pyramidal neurons and volume of CA1 pyramidal layer in 3xTg-AD and wild-type mice treated from 3 to 11 months of age. 3xTg-AD mice displayed CA1 pyramidal layer atrophy that is likely due to reduced neuronal volume because of the absence of neuronal loss. Both lithium treatments of 3xTg-AD mice, which already expressed AD-like pathology, had no effect on CA1 atrophy. However, lithium treatment of wild-type mice, at low (subtherapeutic) doses, induced a significant increase in total CA1 pyramidal neuron number that led to a significant increase in total CA1 pyramidal layer volume. The lithium-induced increase in CA1 neuron number is highly consistent with previous evidence that adult neurogenesis can be exogenously induced in the CA1 pyramidal layer with impact on total CA1 neuron number, thus raising the possibility of the chronic use of low-dose lithium as a strategy to help compensate for neuronal loss in CA1 and perhaps other typically non-neurogenic brain regions in various neurological diseases. With regard to AD, low-dose lithium intervention must be initiated as early as possible in the course of neuropathology for beneficial effects to occur.

A zinc fixative for 3D visualization of cerebral capillaries and pericytes.

BACKGROUND: A large volume of data indicates that disturbances in the morphology and function of the capillary wall may play a causal role in several types of neurodegenerative disorders. We present a highly reproducible staining method for investigating the cerebral capillary network and the pericyte cells within the basement membrane in mice - a specie specific challenging task when uniform staining in thick sections was needed for confocal microscopy or a quantitative analysis, e.g. stereological investigation using 3D probes. NEW METHOD: We perfused C57BL6/Jbom mice and immersion fixated the brains with an aldehyde free zinc fixative, which is normally used for paraffin embedded tissues, and stained for CD31 and Collagen Type IV positive capillaries in 100µm thick sections. RESULTS: Using the milder zinc fixative allowed complete immunohistochemical visualization of the cerebral capillary network in 100µm thick sections using CD31 or Collagen Type IV antibodies. Moreover CD31 or Collagen Type IV staining revealed the presence of pericytes, which was confirmed by a fluorescent co-localization with the NG2 pericyte marker. COMPARISON WITH EXISTING METHODS: Compared with conventional aldehyde-based fixative, this method resulted in a homogeneous staining through the entire depth of thick sections with very limited background staining and well-preserved morphology. CONCLUSIONS: This method is suitable for 3D stereological analysis of capillary networks and pericytes within thick brain sections using CD31 or Collagen Type IV antibodies.

Quantitative analysis of the capillary network of aged APPswe/PS1dE9 transgenic mice.

A combination of immunohistochemical and stereological techniques were used to investigate the capillary network in the cerebral cortex of 18-month-old APPswe/PS1dE9 transgenic (Tg) mice and control littermates. Data regarding total capillary length, segment number, diffusion radius, and pericyte number are presented. The total length was 60 meters and there was a one-to-one relationship between the number of capillary segments and pericytes in both groups. Significant differences were not observed in the Tg and wild-type controls indicating that the Alzheimer's-like amyloidosis produced in this Tg mouse has a minimal affect on the structural integrity of the cerebral capillary network.

What to report: information to be included in the publication of a stereological study.

In publications of stereological studies, descriptions of the method and data are often incomplete. This article discusses how to describe a stereological study in such a way that allows readers and reviewers to access the quality of the data and reproduce the study.

Local estimators of size in stereological studies.

There are two general categories of stereological methods, global and local. Global stereological estimators include estimators of total volume, surface, length, and number. Local estimators can be used to estimate the volumes or surfaces of single objects and, thereby, the distribution of object sizes or the mean size of objects within a population. Two local estimators that have proved to be of practical value for measuring the volume of cells in neurobiological studies are the nucleator and the rotator, which are the focus of this article. Variants of the nucleator can be used for estimating object number, object surface, and the spatial distributions of objects.

Counting and measuring ultrastructural features of biological samples.

Ultrastructural features of cells can be fractions of a micrometer in diameter, and electron microscopy is needed to resolve them to a degree that is compatible with stereological techniques. Because the focal depth of transmission electron microscopy (TEM) images is thousands of times greater than the thickness of the sections used with TEM, virtual sectioning of sections suitable for TEM is not possible, as it is with light microscopy and the optical disector probe. With features the size of neuronal synapses, for example, this necessitates the use of physical sections and physical disectors. Regardless of how the imaging is performed, the design of stereological studies for quantifying ultrastructural features will be essentially the same as that used in the example described here, which uses physically separated ultrathin sections viewed with conventional TEM to estimate the number and size of synapses in a particular brain region.

Optimizing the sampling scheme for a stereological study: how many individuals, sections, and probes should be used.

Stereology provides meaningful quantitative descriptions of the geometry of three-dimensional (3D) structures from measurements that are made on two-dimensional (2D) images. A pilot stereological study will provide information that can be used to rationalize how many individuals, sections, and probes should be used to ensure that one is sampling enough, but not too much, to achieve the goal of a study. This general approach is exemplified in the thought experiment described here, which involves a comparison of the means of estimates of the total number of neurons N in two groups, using the simplest of statistical tests, the Student's t-test. It is also applicable to studies involving other estimates of total quantities such as volume, surface, and length obtained from a parallel series of sections.

Estimating length in biological structures.

Length estimates of particular features of biological tissues can be useful in evaluating function. Such estimates have been notoriously difficult to obtain because of the requirement for an isotropic interaction between the area probes and the linear features of cells and tissues, which are most likely not isotropically oriented. For complex embedded structures, such as subdivisions of the brain, the turning of the tissue before sectioning that is needed to ensure an isotropic interaction has made it difficult to delineate many regions of interest and limited the number of unbiased stereological studies of length. The recent development of a virtual isotropic spherical probe, the spaceball, makes it relatively easy for the isotropic interaction between probe and structure to be realized. This article describes the use of the spaceball probe to estimate length, and gives an example of estimating total capillary length in CA1 stratum radiatum of the human hippocampus.

Getting started in stereology.

Stereology involves sampling structural features in sections of tissue with geometrical probes. This article discusses some practical issues that must be dealt with when getting started in stereology, including tissue preparation methods and determining how many tissue sections and probes are needed to make a stereological estimate.

Tissue shrinkage and stereological studies.

Shrinkage often takes place in biological tissues during the different phases of preparation for microscopy. This can have detrimental effects on the stereological estimates, even when unbiased procedures are used. There are different types of shrinkage, and an awareness of them is essential when designing stereological studies. Some forms of shrinkage can be taken into account to ensure the unbiasedness of an estimator, but some cannot and should be avoided. Dimensional changes that take place during fixation, embedding, sectioning, mounting, and staining can seriously compromise one's ability to make assumption-free estimates of length and surface, but there are steps that can be taken to reduce the impact of these changes on estimates of object number and size. This article describes types of shrinkage and the effects of shrinkage on estimators of object number. It gives an example of how to make a number-weighted correction of section thickness and also discusses the consequences of shrinkage for the validity of estimates of object size.

Estimating surface area in biological structures.

Stereological estimates of surface have led to important insights into normal and pathological processes. This article describes the process of estimating surface area in biological structures. It includes a discussion of relationship equations for estimating surface and procedures for estimating surface area of components of organ systems. It also provides an example of estimation of the area of the pial surface of the human brain.

Isotropy, iSectors, and vertical sections in stereology.

Stereology provides meaningful quantitative descriptions of the geometry of three-dimensional (3D) structures from measurements that are made on two-dimensional (2D) images. One important consideration when designing such studies is that both length and surface features may have preferred orientations in 3D space; that is, they may not be isotropic. To fully understand the global estimators of length L and surface area S, it is essential that one understand the problems that the inherent anisotropy in the structure of most living organisms generates when designing unbiased sampling schemes for estimating length and surface. These same issues also apply to the use of local estimators of object volume v and surface s, in that they use line and surface probes. These problems and the various solutions to them, which involve sectioning and probing tissue in particular ways, are presented in this article.

The capillary dysfunction hypothesis of Alzheimer's disease.

It is widely accepted that hypoperfusion and changes in capillary morphology are involved in the etiopathogenesis of Alzheimer's disease (AD). This is difficult to reconcile with the hyperperfusion observed in young high-risk subjects. Differences in the way cerebral blood flow (CBF) is coupled with the local metabolic needs during different phases of the disease can explain this apparent paradox. This review describes this coupling in terms of a model of cerebral oxygen availability that takes into consideration the heterogeneity of capillary blood flow patterns. The model predicts that moderate increases in heterogeneity requires elevated CBF in order to maintain adequate oxygenation. However, with progressive increases in heterogeneity, the resulting low tissue oxygen tension will require a suppression of CBF in order to maintain tissue metabolism. The observed biphasic nature of CBF responses in preclinical AD and AD is therefore consistent with progressive disturbances of capillary flow patterns. Salient features of the model are discussed in the context of AD pathology along with potential sources of increased capillary flow heterogeneity.

PiB Fails to Map Amyloid Deposits in Cerebral Cortex of Aged Dogs with Canine Cognitive Dysfunction.

Dogs with Canine Cognitive Dysfunction (CCD) accumulate amyloid beta (Aß) in the brain. As the cognitive decline and neuropathology of these old dogs share features with Alzheimer's disease (AD), the relation between Aß and cognitive decline in animal models of cognitive decline is of interest to the understanding of AD. However, the sensitivity of the biomarker Pittsburgh Compound B (PiB) to the presence of Aß in humans and in other mammalian species is in doubt. To test the sensitivity and assess the distribution of Aß in dog brain, we mapped the brains of dogs with signs of CCD (n = 16) and a control group (n = 4) of healthy dogs with radioactively labeled PiB ([(11)C]PiB). Structural magnetic resonance imaging brain scans were obtained from each dog. Tracer washout analysis yielded parametric maps of PiB retention in brain. In the CCD group, dogs had significant retention of [(11)C]PiB in the cerebellum, compared to the cerebral cortex. Retention in the cerebellum is at variance with evidence from brains of humans with AD. To confirm the lack of sensitivity, we stained two dog brains with the immunohistochemical marker 6E10, which is sensitive to the presence of both Aß and Aß precursor protein (AßPP). The 6E10 stain revealed intracellular material positive for Aß or AßPP, or both, in Purkinje cells. The brains of the two groups of dogs did not have significantly different patterns of [(11)C]PiB binding, suggesting that the material detected with 6E10 is AßPP rather than Aß. As the comparison with the histological images revealed no correlation between the [(11)C]PiB and Aß and AßPP deposits in post-mortem brain, the marked intracellular staining implies intracellular involvement of amyloid processing in the dog brain. We conclude that PET maps of [(11)C]PiB retention in brain of dogs with CCD fundamentally differ from the images obtained in most humans with AD.

Systematic versus random sampling in stereological studies.

The sampling that takes place at all levels of an experimental design must be random if the estimate is to be unbiased in a statistical sense. There are two fundamental ways by which one can make a random sample of the sections and positions to be probed on the sections. Using a card-sampling analogy, one can pick any card at all out of a deck of cards. This is referred to as independent random sampling because the sampling of any one card is made without reference to the position of the other cards. The other approach to obtaining a random sample would be to pick a card within a set number of cards and others at equal intervals within the deck. Systematic sampling along one axis of many biological structures is more efficient than random sampling, because most biological structures are not randomly organized. This article discusses the merits of systematic versus random sampling in stereological studies.

Estimating volume in biological structures.

Estimates of volume can be useful in comparative and experimental studies of tissues and organs. In this article, a simple procedure for making unbiased estimates of total volume using Cavalieri's principle is described. This is followed by a description of how point probes can be used to make unbiased estimates of the areas of sectional profiles and how, when combined with Cavalieri's principle, point counting can be used to make an unbiased estimate of the volume of a structural feature.