Vibratome Sections Research Articles

Morphology of the brain in Hutchinsoniella macracantha (Cephalocarida, Crustacea)

External morphological features of Cephalocarida have long been interpreted as plesiomorphic with regard to those of other crustaceans. Based on transmission electron microscopy and light microscopy, however, the brain in the cephalocarid Hutchinsoniella macracantha has been shown to contain a number of structures that are more difficult to interpret in an evolutionary context. These include the multi-lobed complex, a unique cluster of neuropils associated with the olfactory lobes. To establish a well-founded comparison of phylogenetically relevant, neuroanatomical data from Cephalocarida to other arthropods, we investigated the brain in H. macracantha using immunolabeling (acetylated α-tubulin, serotonin, RFamide, histamine) and nuclear counter stains of whole mounts and vibratome sections analyzing specimens with confocal laser scanning microscopy and computer-aided 3D-reconstruction. Other 3D-reconstructions were based on serial 1 μm semi-thin sections. The multi-lobed complex features a pedunculus and shows detailed homologies with the mushroom bodies of certain Insecta and Lithobiomorpha (Chilopoda), suggesting that the hemiellipsoid bodies in Remipedia and Malacostraca have derived from a cephalocarid-like pattern. Like the corresponding tracts in Insecta, the olfactory globular tracts linking the multi-lobed complex to the olfactory lobes are ipsilateral, probably constituting the plesiomorphic pattern from which the decussating tracts in Remipedia and Malacostraca have evolved. The olfactory lobes in H. macracantha are uniquely organized into vertical stacks of olfactory glomeruli whose exact shape could not be identified. Similarly to Malacostraca and Insecta, the olfactory glomeruli in H. macracantha are innervated by serotonin-like, RFamide-like, and histamine-like immunoreactive interneurons. This suggests homology of the olfactory lobes across Tetraconata, despite the different morphological organization. Although H. macracantha lacks elongated, unpaired midline neuropils known from the protocerebrum of other Arthropoda, the possible rudiment of a central-body-like neuropil that receives decussating fibers from anterior somata was revealed by the serotonin-like immunoreactive pattern.

Estandarización de un protocolo para la combinación de técnicas neurohistológicas en cortes obtenidos con vibrátomo

The histological study of the nervous system requires the use of special techniques. Currently, no methods are available to visualize simultaneously all the cellular constituents of nervous tissue.A protocol was adapted for staining nervous tissue by modification of a formerly difficult procedure.Slices of brain and spinal cord, 4 mm thick, were taken from adult mice, previously fixed by intracardiac perfusion with 4% paraformaldehyde. Vibratome sections were obtained with thickness of 15-25 µm. These were mounted on glass slides prepared with gelatin as an adhesive. The preparations were subjected to staining protocol Luxol Fast Blue-PAS-hematoxylin (LPH) combined with silver staining method (LPH-Holmes).LPH technique yielded an excellent differentiation of gray and white matter in all regions of the nervous system. A panoramic view of the gray matter was colored pink in contrast to the myelinated nerve fibers and tracts which were light blue. The combination LPH-Holmes retained the staining characteristics but significantly improved the demarcation of axons and tracts.A protocol was standardized for the LPH and LPH-Holmes nervous tissue stains applied in combination to tissue slices obtained with a vibratome. The method was shorter, less wasteful and less expensive than the original and also better preserved the integrity of nervous tissue.

Silver-enhanced immunogold scanning electron microscopy using vibratome sections of rat kidneys: detection of albumin filtration and reabsorption

To show the three-dimensional distribution of proteins in renal cells, we applied the immunogold scanning electron microscopy method using vibratome slices. Kidney specimens from puromycin aminonucleoside (PAN) nephrotic rats and controls were obtained after intravenous infusion of human serum albumin and fixed in periodate-lysine-paraformaldehyde solution. Vibratome slices were incubated with anti-human albumin antibody and 25-nm gold-labeled secondary antibody. After silver enhancement, the immunogold particles were clearly observed by backscatter electron imaging, whereas they were ambiguous in the secondary electron image. The images showed a higher resolution of the tissues at an acceleration voltage of 5 mV than at 10 mV. Immunogold-labeled albumin was observed in the lumen and endocytotic vesicles of the proximal tubules, and on the podocyte surface in the PAN nephrotic rats, whereas only a few particles were observed in the controls. In conclusion, silver-enhanced immunogold scanning electron microscopy at low acceleration voltages using vibratome sections can be applicable for detecting the intracellular/extracellular localization of molecules in solid tissues. We succeeded in visualizing the enhanced albumin endocytosis of the proximal tubules and the exocytosis of albumin from podocytes in the nephrotic rats.

Increased Glucocorticoid Receptor Expression and Activity Mediate the LPS Resistance of SPRET/EI Mice

SPRET/Ei mice are extremely resistant to acute LPS-induced lethal inflammation when compared with C57BL/6. We found that in vivo SPRET/Ei mice exhibit strongly reduced expression levels of cytokines and chemokines. To investigate the role of the potent anti-inflammatory glucocorticoid receptor (GR) in the SPRET/Ei phenotype, mice were treated with the GR antagonist RU486 or bilateral adrenalectomy. Under such conditions, both C57BL/6 and SPRET/Ei mice were strongly sensitized to LPS, and the differences in LPS response between SPRET/Ei and C57BL/6 mice were completely gone. These results underscore the central role of GR in the LPS hyporesponsiveness of SPRET/Ei mice. Compared with C57BL/6, SPRET/Ei mice were found to express higher GR levels, which were reflected in increased GR transactivation. Using a backcross mapping strategy, we demonstrate that the high GR transcription levels are linked to the Nr3c1 (GR) locus on chromosome 18 itself. Unexpectedly, SPRET/Ei mice exhibit a basal overactivation of the hypothalamic-pituitary-adrenal axis, namely strongly increased corticosterone levels, ACTH levels, and adrenocortical size. As a consequence of the excess of circulating glucocorticoids (GCs), levels of hepatic gluconeogenic enzymes are increased, and insulin secretion from pancreatic β-cells is impaired, both of which result in hyperglycemia and glucose intolerance in SPRET/Ei mice. We conclude that SPRET/Ei mice are unique as they display an unusual combination of elevated GR expression and increased endogenous GC levels. Hence, these mice provide a new and powerful tool for the study of GR- and GC-mediated mechanisms, including immune repressive functions, neuroendocrine regulation, insulin secretion, and carbohydrate metabolism.

Evaluating Corneal Collagen Organization Using High-Resolution Nonlinear Optical Macroscopy

Recent developments in nonlinear optical (NLO) imaging using femtosecond lasers provides a noninvasive method for detecting collagen fibers by imaging second harmonic-generated (SHG) signals. However, this technique is limited by the small field of view necessary to generate SHG signals. The purpose of this report is to review our efforts to greatly extend the field of view to assess the entire collagen structure using high-resolution macroscopic (HRMac) imaging. Intact human eyes were fixed under pressure, and the whole cornea (13-mm diameter) was excised and embedded in low-melting point agar for vibratome sectioning (200-300 microm). Sections were then optically scanned using a Zeiss LSM 510 Meta and Chameleon femtosecond laser (Carl Zeiss Microimaging Inc., Thornwood, NY) to generate SHG images. For each vibratome section, an overlapping series of three-dimensional data sets (466 x 466 x 150 microm) were taken, covering the entire tissue (15 mm x 6 mm area) using a motorized, mechanical stage. The three-dimensional data sets were then concatenated to generate an NLO-based tomograph. The HRMac of the cornea yielded large macroscopic (80 megapixels per plane), three-dimensional tomographs with high resolution (0.81 microm lateral, 2.0 microm axial) in which individual collagen fibers (stromal lamellae) could be traced, segmented, and extracted. Three-dimensional reconstructions suggested that the anterior cornea comprises highly intertwined lamellae that insert into the anterior limiting lamina (Bowman's layer). We conclude that HRMac using NLO-based tomography provides a powerful new tool to assess collagen structural organization within the cornea.

Cellular Source of Apolipoprotein E4 Determines Neuronal Susceptibility to Excitotoxic Injury in Transgenic Mice

The lipid transport protein apolipoprotein E (apoE) is abundantly expressed in the brain. Its main isoforms in humans are apoE2, apoE3, and apoE4. ApoE4 is the major known genetic risk factor for Alzheimer's disease and also contributes to the pathogenesis of various other neurological conditions. In the central nervous system, apoE is synthesized by glial cells and neurons, but it is unclear whether the cellular source affects its biological activities. To address this issue, we induced excitotoxic injury by systemic kainic acid injection in transgenic Apoe knockout mice expressing human apoE isoforms in astrocytes or neurons. Regardless of its cellular source, apoE3 expression protected neuronal synapses and dendrites against the excitotoxicity seen in apoE-deficient mice. Astrocyte-derived apoE4, which has previously been shown to have detrimental effects in vitro, was as excitoprotective as apoE3 in vivo. In contrast, neuronal expression of apoE4 was not protective and resulted in loss of cortical neurons after excitotoxic challenge, indicating that neuronal apoE4 promotes excitotoxic cell death. Thus, an imbalance between astrocytic (excitoprotective) and neuronal (neurotoxic) apoE4 expression may increase susceptibility to diverse neurological diseases involving excitotoxic mechanisms.

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Labeling and imaging cells in the zebrafish hindbrain.

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells (1) . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos (2). The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

A Highly Toxic Cellular Prion Protein Induces a Novel, Nonapoptotic Form of Neuronal Death

Several different deletions within the N-terminal tail of the prion protein (PrP) induce massive neuronal death when expressed in transgenic mice. This toxicity is dose-dependently suppressed by coexpression of full-length PrP, suggesting that it results from subversion of a normal physiological activity of cellular PrP. We performed a combined biochemical and morphological analysis of Tg(DeltaCR) mice, which express PrP carrying a 21-aa deletion (residues 105-125) within a highly conserved region of the protein. Death of cerebellar granule neurons in Tg(DeltaCR) mice is not accompanied by activation of either caspase-3 or caspase-8 or by increased levels of the autophagy marker, LC3-II. In electron micrographs, degenerating granule neurons displayed a unique morphology characterized by heterogeneous condensation of the nuclear matrix without formation of discrete chromatin masses typical of neuronal apoptosis. Our data demonstrate that perturbations in PrP functional activity induce a novel, nonapoptotic, nonautophagic form of neuronal death whose morphological features are reminiscent of those associated with excitotoxic stress.

Localization of BDNF mRNA with the Huntington's disease protein in rat brain

BackgroundStudies have implicated reduced levels of brain-derived neurotrophic factor (BDNF) in the pathogenesis of Huntington's disease. Mutant huntingtin (Htt) protein was previously reported to decrease BDNF gene transcription and axonal transport of BDNF. We recently showed that wild-type Htt is associated with the Argonaute 2 microRNA-processing enzyme involved in gene silencing. In dendrites, Htt co-localizes with components of neuronal granules and mRNAs, indicating that it might play a role in post-transcriptional processing/transport of dendritic mRNAs.ResultsWe conducted imaging experiments in cultured cortical neurons to demonstrate the co-localization of endogenous Htt and BDNF mRNA in fixed cells, and co-trafficking of BDNF 3'UTR mRNA with endogenous and fluorescently tagged Htt in live neurons. We used an enhanced technique that combines FISH and immunofluorescent staining to co-localize BDNF mRNA with Htt, Ago2, CPEB and dynein in thick vibratome sections of the rat cortex.ConclusionsIn cultured neurons and sections of the rat cortex, we found BDNF mRNA associated with Htt and components of neuronal RNA granules, which are centers for regulating RNA transport and local translation. Htt may play a role in post-transcriptional transport/targeting of mRNA for BDNF, thus contributing to neurotrophic support and neuron survival.

Relationship between Bunina bodies and TDP‐43 inclusions in spinal anterior horn in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is characterized by upper and lower motor neurone involvement with Bunina bodies (BBs) and TDP-43 inclusions. To elucidate the relationship between BBs and TDP-43 inclusions, we examined the spinal cord from 18 patients with ALS. Five serial sections from lumbar cord were first stained with haematoxylin and eosin to detect BBs and subsequently immunostained with anti-TDP-43 antibody. Immunoelectron microscopy was performed on vibratome sections from two cases of ALS. BBs were found in 15 out of 18 cases. TDP-43 inclusions were found in all the cases. The average incidence of anterior horn cells with BBs and TDP-43 inclusions relative to the total number of neurones was 17.1% and 46.4%, respectively. The concurrence of both inclusions in the same neurones was found in 15 cases. The incidence of co-localization of BBs and TDP-43 inclusions was 15.7% of total neurones. The frequency of TDP-43 inclusions was significantly higher in neurones with BBs than in those without. Ultrastructurally, TDP-43-immunoreactive filamentous structures were intermingled with early-stage BBs, but not associated with advanced-stage BBs. These findings suggest that there is a close relationship in the occurrence between BBs and TDP-43 inclusions.

Preparation of Fixed Xenopus Embryos for Confocal Imaging

Although live imaging of embryonic development is a powerful approach, it is often essential to use immunostaining or in situ hybridization to reveal structures or to localize proteins or gene expression patterns. For these approaches, fixed embryos must be used, and Xenopus offers many advantages. Xenopus is a vertebrate tetrapod closely related to mammals. The large size of their embryos makes them ideal for imaging patterns of gene expression during development. Also, individual Xenopus embryonic cells are larger than those of other vertebrate models, making them suitable for imaging cell morphology and subcellular processes. Xenopus embryos are amenable to simple manipulations of gene function, including knockdown and misexpression, and the large numbers of embryos produced allow even an inexperienced researcher to perform such manipulations on hundreds of embryos per day. Transgenesis is quite effective as well. Finally, because the fate map of Xenopus embryos is stereotypical, simple targeted microinjections can reliably deliver reagents into specific tissues and cell types for gene manipulation or for imaging. To improve image quality for fixed specimens, the pigment of the Xenopus embryo can be removed by bleaching. Also, although Xenopus embryos are opaque because of the large amounts of yolk stored in each cell, they can easily be rendered transparent using a process called clearing. This protocol describes the methods for bleaching and clearing Xenopus embryos, as well as a simple procedure for vibratome sectioning. These approaches are effective for imaging embryos and cells following immunostaining or in situ hybridization.

Analysis of Gene Expression by In Situ Hybridization on Adult Zebrafish Brain Sections

INTRODUCTIONWhole-mount in situ hybridization is a powerful method for the analysis of gene expression in zebrafish. However, the thickness of adult brain tissue often hampers penetration of reagents, making the direct application of protocols normally used for embryonic stages on adult tissues difficult. This protocol presents a detailed procedure for the dissection, preparation, and processing of the adult zebrafish brain for detection of gene expression in situ using digoxigenin (DIG)-labeled RNA probes on vibratome sections: The entire procedure is presented, including synthesis of labeled antisense RNA, sectioning of the adult zebrafish brain, and in situ hybridization. Using this method, all parts of the tissue are easily labeled and expression patterns are reproduced faithfully between trials. Unlike whole-mount preparations, gene expression examined on vibratome sections can easily be compared to a detailed zebrafish brain atlas and assigned to specific tissues.

ENDOCRINE ORGANS AND LASER SCANNING CONFOCAL MICROSCOPY (LSCM) IMAGING: VASCULAR BED IN HUMAN SPLEEN

This work was aimed to utilize the precise method of laser confocal microscopy (LSCM) to depict the image of spatial relationships of the vessel network in the tissue structures of the human spleen. With the use of serial paraffin or vibratome sections of more than 20 μm thickness infiltrated with eosin fluorescence dye the images of arterial and venous walls of different calibres, capillaries, and venous sinuses were morphologically revealed. Venous sinuses were frequently found to create mutually communicating branches and their lining projected into the lumen protruding cells with distinct spherically or ovally shaped nuclei, positioned on the brightly fluorescent and fragmented lamina basalis. The presence of lymphocytes was distinct in periarteriolar lymphoid sheath (PALS) and lymphatic follicles. Lining cells of the red pulp veins sporadically contained marked eosinophilic granules. The method of LSCM allowed: 1. to reveal two-dimensional and sharp image of the human spleen structures, 2. to investigate the vertical course of venous structures in the tissue, 3. to obtain serial optic sections in z axis to their maximum spatial projections. These data will also serve for the creation of three-dimensional images of vessel network in the human spleen in the future studies.

Combinations of genetic mutations in the adult neural stem cell compartment determine brain tumour phenotypes

It has been suggested that intrinsic brain tumours originate from a neural stem/progenitor cell population in the subventricular zone of the post-natal brain. However, the influence of the initial genetic mutation on the phenotype as well as the contribution of mature astrocytes to the formation of brain tumours is still not understood. We deleted Rb/p53, Rb/p53/PTEN or PTEN/p53 in adult subventricular stem cells; in ectopically neurografted stem cells; in mature parenchymal astrocytes and in transplanted astrocytes. We found that only stem cells, but not astrocytes, gave rise to brain tumours, independent of their location. This suggests a cell autonomous mechanism that enables stem cells to generate brain tumours, whereas mature astrocytes do not form brain tumours in adults. Recombination of PTEN/p53 gave rise to gliomas whereas deletion of Rb/p53 or Rb/p53/PTEN generated primitive neuroectodermal tumours (PNET), indicating an important role of an initial Rb loss in driving the PNET phenotype. Our study underlines an important role of stem cells and the relevance of initial genetic mutations in the pathogenesis and phenotype of brain tumours.

Retrogradely transported fluorogold accumulates in lysosomes of neurons and is detectable ultrastructurally using post-embedding immuno-gold methods

For ultrastructural studies, it is of great interest to be able to combine anatomical tracer techniques with sensitive immunohistochemical methods. Fluorogold (FG) is a fluorescent and retrogradely transported anatomical tracer, which is commonly used to label neurons in the brain and spinal cord for light microscopic studies. We here describe a method for detecting FG-labeled somata in the electron microscope using a high resolution post-embedding immuno-gold method. For this purpose, spinal motoneurons were retrogradely labeled by an intraperitoneal injection of FG in the adult rat. The rats were intravascularly perfused with a fixative solution containing 2% paraformaldehyde and 1–2% glutaraldehyde. Vibratome sections of spinal cord tissues were cryo-protected in glycerol, freeze substituted in methanol containing uranyl acetate, and embedded in the Lowicryl HM20 resin at low temperatures. Electron microscopic analysis demonstrated atypical lysosome-like structures in the cytoplasm of FG-labeled motoneurons. Subsequent post-embedding immuno-gold labeling demonstrated prominent accumulation of FG in numerous lysosomes but not in other organelles or cytoplasmic compartments of the labeled neurons. The protocol is versatile and allows for combining anatomical tracing of neurons with, e.g., neuro-transmitter studies in the electron microscope. We suggest that the described method for sensitive detection of FG in the spinal cord may also have broad applicability to other areas of the central nervous system.

Identification of hypophysiotropic luteinizing hormone-releasing hormone (LHRH) neurons by combined retrograde labeling and immunocytochemistry.

The decapeptide luteinizing hormone-releasing hormone (LHRH) is produced by telencephalic and diencephalic neurons and transported to the median eminence (ME). After having been released from nerve terminals, it is carried by the hypophysial portal vessels to the anterior pituitary, where it stimulates the production and release of luteinizing hormone (LH) and follicle stimulating hormone (FSH). Those LHRH neurons which project to the ME represent the final common pathway for the regulation of the pituitary/gonadal axis. We identified these neurons by injecting a retrograde tracer, the lectin wheat germ agglutinin (WGA), into the external zone of the ME. Eight to 24 hours later colchicine was given into the lateral ventricle and 24-48 hours after the WGA injection the animals were sacrificed. Vibratome sections of the brains were stained simultaneously for WGA and LHRH with a dual immunocytochemical technique. Approximately 70% of the LHRH neurons in the septum and the anterior hypothalamus were double-labeled, indicating that they projected to the ME. Double labeled LHRH cells were either smooth, fusiform or "spiny". WGA-accumulating LHRH perikarya were intermixed with single-labeled LHRH cells. The remaining 30% of the LHRH neurons which were not labeled with WGA appeared to project to different hypothalamic and extrahypothalamic areas of the brain. Our results suggest that there are at least two populations of LHRH neurons, one with access to the portal capillaries of the ME and functionally related to the regulation of the pituitary, and one without access to capillaries of the ME, functionally probably related to intracerebral neurotransmission or modulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Lycopersicon esculentum lectin: an effective and versatile endothelial marker of normal and tumoral blood vessels in the central nervous system

The binding of Lycopersicon esculentum lectin (LEA) to the vascular endothelium was studied in the central nervous system of rat, mouse and guinea pig at different developmental ages, and in a gliosarcoma model. Our observations showed that LEA consistently stained the entire vascular tree in the spinal cord and in the brain of all animal species at all developmental ages investigated. In the tumor model, the staining of the vascular network was very reproducible, enabled an easy identification of vascular profiles and displayed a higher efficiency when compared to two other commonly used vascular marker (EHS laminin and PECAM-1). Moreover, our results showed that LEA staining was comparable in both vibratome and paraffin sections and could be easily combined with other markers in double labeling experiments. These observations indicate that LEA staining may represent an effective and versatile endothelial marker for the study of the vasculature of the central nervous system in different animal species and experimental conditions.

IN VITRO INDUCTION OF AUTOTETRAPLOID ACTINIDIA PLANTS AND THEIR FIELD EVALUATION FOR CROP IMPROVEMENT

IN VITRO INDUCTION OF AUTOTETRAPLOID ACTINIDIA PLANTS AND THEIR FIELD EVALUATION FOR CROP IMPROVEMENT

Remodeling of the Corticospinal Innervation and Spontaneous Behavioral Recovery After Ischemic Stroke in Adult Mice

To elucidate how the motor pathways rewire the denervated tissue after stroke, we investigated remodeling of the corticospinal tract (CST) in transgenic mice with yellow fluorescent protein CST labeling in conjunction with transsynaptic pseudorabies virus retrograde tracing. Adult male CST-yellow fluorescent protein mice were subjected to permanent right middle cerebral artery occlusion (n=8/group). Foot-fault test was performed to monitor functional deficit and recovery. Pseudorabies virus tracer was injected into the left forelimb muscles at 1 or 4 weeks after middle cerebral artery occlusion (4 days before euthanasia), respectively. A third group of CST-yellow fluorescent protein mice without middle cerebral artery occlusion was used for normal control (n=6). The yellow fluorescent protein labeling of CST in the cervical cord and pseudorabies virus labeling of pyramidal neurons in the bilateral cortices were measured on vibratome sections using a confocal imaging system. Compared with normal animals, axonal density in the stroke-affected side of the cervical cord was significantly decreased at 11 days (P<0.001) and significantly increased at 32 days after stroke compared with the Day 11 values (P<0.05). Pseudorabies virus labeling was significantly decreased in the ischemic hemisphere 11 days after middle cerebral artery occlusion (P<0.001). In contrast, a significant increase was observed in pseudorabies virus labeling of bilateral cortices 32 days after stroke compared with 11 days (P<0.05). The CST axonal density in the denervated spinal cord and pyramidal neuron labeling in the bilateral cortices were significantly correlated with behavioral recovery (P<0.05). Spontaneous functional recovery after stroke may, at least in part, be attributed to neuronal remodeling in the corticospinal system.