Fiber Tracing Research Articles

Quantification of Nucleosome Stacking in Single 30 nm Chromatin Fibers

DNA and core histones form dense 30 nm chromatin fibers in vitro. The order of nucleosome stacking drives the folding of 30 nm fibers and may control enzymatic accessibility of the linker DNA in vivo. The energy and dynamics of nucleosome stacking are not well quantified. Here, we investigated nucleosome stacking by pulling on reconstituted chromatin fibers with magnetic tweezers. The force extension traces of fibers are well described as transition between a Hookean spring representing the 30 nm fiber and a worm like chain representing a bead-on-a string conformation. The results show that nucleosome stacking is reversible and force dependent. We measure a stacking energy of 17kBT and an unstacking distance that indicates the full exposure of the linker DNA. The time traces at constant force between 3.5 pN and 6.0 pN show that multiple nucleosome unstacking and restacking events take place simultaneously and non-cooperatively. The salt dependence of unstacking suggests competition between monovalent ions and divalent ions. These experiments provide the first single molecule data on nucleosome stacking and define a dynamic framework for chromatin organization in higher order structures.

Diffusion tensor magnetic resonance imaging of glial brain tumors

Aim To evaluate the author's experience with the use of diffusion tensor magnetic resonance imaging (DTI) on patients with glial tumors. Methods A retrospective evaluation of a group of 24 patients with glial tumors was performed. There were eight patients with Grade II, eight patients with Grade III and eight patients with Grade IV tumors with a histologically proven diagnosis. All the patients underwent routine imaging including T2 weighted images, multidirectional diffusion weighted imaging (measured in 60 non-collinear directions) and T1 weighted non-enhanced and contrast enhanced images. The imaging sequence and evaluation software were produced by Massachusetts General Hospital Corporation (Boston, MA, USA). Fractional anisotropy (FA) maps were calculated in all patients. The white matter FA changes were assessed within the tumorous tissue, on the tumorous borderline and in the normally appearing white matter adjacent to the tumor. A three-dimensional model of the white matter tract was created to demonstrate the space relationship of the tumor and the capsula interna or corpus callosum in each case using the following fiber tracing parameters: FA step 0.25 and a tensor declination angle of 45 gr. An additional assessment of the tumorous tissue enhancement was performed. Results A uniform homogenous structure with sharp demargination of the Grade II tumors and the wide rim of the intermedial FA in all Grade III tumors respectively, were found during the evaluation of the FA maps. In Grade IV tumors a variable demargination was noted on the FA maps. The sensitivity and specificity for the discrimination of low- and high-grade glial tumors using FA maps was revealed to be 81% and 87% respectively. If the evaluation of the contrast enhancement was combined with the evaluation of the FA maps, both sensitivity and specificity were 100%. Conclusion Although the evaluation of the fractional anisotropy maps is not sufficient for glioma grading, the combination of the contrast enhancement pattern and fractional anisotropy maps evaluation improves the possibility of distinguishing low- and high-grade glial tumors. Three-dimensional models of the white matter fibers in the corpus callosum and the internal capsule may be used in the presurgical planning.

Neural tractography using an unscented Kalman filter.

We describe a technique to simultaneously estimate a local neural fiber model and trace out its path. Existing techniques estimate the local fiber orientation at each voxel independently so there is no running knowledge of confidence in the estimated fiber model. We formulate fiber tracking as recursive estimation: at each step of tracing the fiber, the current estimate is guided by the previous. To do this we model the signal as a mixture of Gaussian tensors and perform tractography within a filter framework. Starting from a seed point, each fiber is traced to its termination using an unscented Kalman filter to simultaneously fit the local model and propagate in the most consistent direction. Despite the presence of noise and uncertainty, this provides a causal estimate of the local structure at each point along the fiber. Synthetic experiments demonstrate that this approach reduces signal reconstruction error and significantly improves the angular resolution at crossings and branchings. In vivo experiments confirm the ability to trace out fibers in areas known to contain such crossing and branching while providing inherent path regularization.

Tracing retinal fiber trajectory with a method of transposon-mediated genomic integration in chick embryo

We report convenient retinal fiber tracing by transfecting the tracer cDNA by in ovo electroporation. Long-term and stable expression of tracer proteins such as green fluorescent protein is achieved by transposon-mediated genome integration of the tracer protein expression cassette. We carried out coelectroporation of a plasmid containing CAGGS-tracer cDNA flanked by the Tol2 transposable element along with a transposase expression vector to the optic vesicle of chick embryos at stage 11. By selecting electrodes, we can label a large group of retinal ganglion cells, or a small group of retinal ganglion cells; parallel electrodes assure transfection of large areas of the retina, and needle type electrodes label small areas of the retina. The retinal fiber trajectory and terminal zone (TZ) could be detected in the precise retinotopic manner on the contra-lateral side of the optic tectum. The method has advantage in that we can show the retinal fiber trajectory in relation to the molecules that are responsible for pathfinding for the retinal fibers in the same specimen.

DESARROLLO Y PUESTA EN MARCHA DE SOFTWARE DE TRACTOGRAFIA

Abstract: Diffusion weighted MRI can measure the random motion of water molecules in biological tissue. These motions are captured using magnetic gradients that dephase the precession of water molecules that move along the direction of the gradient. Dephasings show up as small attenuations in signal intensity.This information can be used in tissues such as muscles, spine, medulla and white matter to measure the ani-sotropy and assess fiber integrity. In this work, a new software for the post-processing of diffusion weighted MRI is presented.The software can read datasets from a variety of scanners. Diffusion is modeled using ellipsoids that are represented mathematically by means of a tensor, estimated from entry dataset. Various indices such as diffusion tensor eigenvalues, fractional anisotropy, types of anisotropy, mean diffu-sivity, and principal directions are computed. Results are interactively visualized by using axial planes or a three-dimensional approach. The software includes a nerve fiber tracing module. This tool works on PC-based workstations through a graphical user interface or by using the command line.

In VivoTracing of Neural Tracts in the Intact and Injured Spinal Cord of Marmosets by Diffusion Tensor Tractography

In spinal cord injury, axonal disruption results in motor and sensory function impairment. The evaluation of axonal fibers is essential to assess the severity of injury and efficacy of any treatment protocol, but conventional methods such as tracer injection in brain parenchyma are highly invasive and require histological evaluation, precluding clinical applications. Previous advances in magnetic resonance imaging technology have led to the development of diffusion tensor tractography (DTT) as a potential modality to perform in vivo tracing of axonal fibers. The properties and clinical applications of DTT in the brain have been reported, but technical difficulties have limited DTT studies of the spinal cord. In this study, we report the effective use of DTT to visualize both intact and surgically disrupted spinal long tracts in adult common marmosets. To verify the feasibility of spinal cord DTT, we first performed DTT of postmortem marmosets. DTT clearly illustrated spinal projections such as the corticospinal tract and afferent fibers in control animals, and depicted the severed long tracts in the injured animals. Histology of the spinal cords in both control and injured groups were consistent with DTT findings, verifying the accuracy of DTT. We also conducted DTT in live marmosets and demonstrated that DTT can be performed in live animals to reveal in vivo nerve fiber tracing images, providing an essential tool to evaluate axonal conditions in the injured spinal cord. Taken together, these findings demonstrate the feasibility of applying DTT to preclinical and clinical studies of spinal cord injury.

Comparison of fiber tracts derived from in-vivo DTI tractography with 3D histological neural tract tracer reconstruction on a macaque brain

Since the introduction of diffusion weighted imaging (DWI) as a method for examining neural connectivity, its accuracy has not been formally evaluated. In this study, we directly compared connections that were visualized using injected neural tract tracers (WGA-HRP) with those obtained using in-vivo diffusion tensor imaging (DTI) tractography. First, we injected the tracer at multiple sites in the brain of a macaque monkey; second, we reconstructed the histological sections of the labeled fiber tracts in 3D; third, we segmented and registered the fibers (somatosensory and motor tracts) with the anatomical in-vivo MRI from the same animal; and last, we conducted fiber tracing along the same pathways on the DTI data using a classical diffusion tracing technique with the injection sites as seeds. To evaluate the performance of DTI fiber tracing, we compared the fibers derived from the DTI tractography with those segmented from the histology. We also studied the influence of the parameters controlling the tractography by comparing Dice superimposition coefficients between histology and DTI segmentations. While there was generally good visual agreement between the two methods, our quantitative comparisons reveal certain limitations of DTI tractography, particularly for regions at remote locations from seeds. We have thus demonstrated the importance of appropriate settings for realistic tractography results.

Delayed Transplantation with Exogenous Neurotrophin Administration Enhances Plasticity of Corticofugal Projections after Spinal Cord Injury

Functional deficits following spinal cord injury (SCI) result from a disruption of corticofugal projections at the lesion site. Not only direct regeneration of the severed axons but also anatomical re-organization of spared corticofugal pathways can reestablish connections between the supraspinal and spinal motor centers. We have previously shown that delayed transplantation of fetal spinal cord tissue and neurotrophin administration by two weeks after SCI supported recovery of forelimb function in adult rats. The current study determined whether the same intervention enhances plasticity of corticofugal fibers at the midbrain and spinal cord level. Anterograde tracing of the left corticorubral fibers revealed that the animals with transplants and neurotrophins (BDNF or NT-3) increased the extent of the traced fibers crossing to the right red nucleus (RN), of which the axons are spared by a right cervical overhemisection lesion. More neurons in the left motor cortex were recruited by the treatment to establish connections with the right RN. The right corticorubral projections also increased the density of midline crossing fibers to the axotomized left RN in response to transplants and neurotrophins. Transplants plus NT-3, but not BDNF, significantly increased the amount of spared corticospinal fibers in the left dorsolateral funiculus at the spinal level both rostral and caudal to the lesion. These results suggest that corticofugal projections retain the capacity until at least two weeks after injury to undergo extensive reorganization along the entire neuraxis in response to transplants and neurotrophins. Targeting anatomical plasticity of corticofugal projections may be a promising strategy to enhance functional recovery following incomplete SCI.

Caracterização físico-química de okara e aplicação em pães do tipo francês

A composição química do extrato aquoso de soja e de seu subproduto okara foi determinada. Em base seca, para o okara, o teor dos componentes encontrados foi de 2,8% de cinzas, 37% de proteínas, 13% de lipídios, 42,5% de fibras alimentares e 4,7% de carboidratos solúveis. Para o extrato aquoso esses valores foram de 2,4% de cinzas, 33% de proteínas, 17,7% de lipídios, 41,4% de carboidratos solúveis e traços de fibras alimentares. Pães do tipo francês foram adicionados de 0, 5, 10 e 15% de okara seco. Foi realizada a análise sensorial por meio do teste de preferência que demonstrou que os pães contendo 5% e 10% de okara tiveram níveis de aceitação iguais e superiores àqueles contendo 15%. Assim, os pães contendo 10% de okara foram submetidos ao teste sensorial de intenção de compra, revelando média 4,2 numa escala de 1 a 5, ou seja, uma avaliação favorável do ponto de vista comercial. Pães adicionados de 5% e 10% de okara podem ser denominados produtos "fonte" e "ricos" em fibras, respectivamente.

Spatial resolution dependence of DTI tractography in human occipito-callosal region

Diffusion tensor imaging (DTI) and fiber tracking have been used to measure the fiber structural connectivity in humans in a non-invasive manner. However, low sensitivity is a principal limitation of these methods, causing a large number of possibly missing fiber tracts (FTs). Here we studied how the spatial resolution affects the sensitivity of the fiber tracing by rescaling data to different resolutions. Our data suggest that the spatial resolution can change the degree of the asymmetric cross-callosal connections in the lower visual field (loVF) compared to the upper visual field (upVF). Among connections from loVF, a larger voxel size resulted in a smaller number of FTs that was not commensurate to the number of seed points, while the number of connections from upVF was not significantly affected by variation in seeding point numbers. We conclude from our study that the spatial resolution of the acquired data will have to be taken into consideration in interpreting DTI fiber tracking data. Our results further suggest that the acquisition resolution of around 2 mm iso-voxel in the conventional DTI scheme can reconstruct the asymmetric upper and lower white matter structure in occipito-callosal region.

Synapses formed by normal and abnormal hippocampal mossy fibers

The axon terminals (mossy fibers) of hippocampal dentate granule cells form characteristic synaptic connections with large spines or excrescences of both hilar mossy cells and CA3 pyramidal neurons. Interneurons of the hilar region and area CA3 are also prominent targets of mossy fibers. The tracing of biocytin-filled mossy fibers and immunolabeling of target cells with interneuron markers has revealed that the majority of mossy fiber synapses project to gamma aminobutyric acid (GABA)-ergic inhibitory interneurons rather than to excitatory principal cells, although the functional implications of these quantitative differences are unclear. Following a brief description of the "classical" mossy fiber synapse on excrescences of CA3 pyramidal cells, the present review focuses on the contacts formed between granule cells and GABAergic interneurons, both normally and after synaptic reorganization. In response to deafferentation of mossy cell target cells, which include both granule cells and interneurons, mossy fibers "sprout" new axon collaterals that form a band of supragranular mossy fibers in the inner molecular layer of the dentate gyrus. Although most newly formed recurrent mossy fibers establish synapses with granule cells, there is an apparently convergent input of new mossy fibers onto GABA-immunoreactive interneuron dendrites that traverse the inner molecular layer. These mossy fiber-interneuron synapses in the dentate gyrus are observed in chronically epileptic rats and may be the structural correlate of the granule cell hyperinhibition observed in these animals in vivo. Together, the findings reviewed here establish mossy fiber synapses as an important component of inhibitory circuits in the hippocampus.

Phase-matched four-wave mixing and sensing of water molecules by coherent anti-Stokes Raman scattering in large-core-area hollow photonic-crystal fibers

Phase-matched four-wave mixing is demonstrated for millijoule nanosecond pulses guided by photonic bandgaps of hollow fibers with a two-dimensionally periodic cladding and a core diameter of ∼50µm. Raman resonances related to the stretching vibrations of water molecules inside the hollow fiber core are detected in the spectrum of the four-wave mixing signal, suggesting phase-matched coherent anti-Stokes Raman scattering in hollow photonic-crystal fibers as a convenient sensing technique for condensed-phase species adsorbed on the inner fiber walls and trace gas detection.

Comparison of the Bioactive Compounds and Antioxidant Potentials of Fresh and Cooked Polish, Ukrainian, and Israeli Garlic

Garlic (Allium sativum L.) is an essential part of Polish, Ukrainian, and Israeli cuisine. The aim of this investigation was to compare the changes in bioactive compounds, proteins, and antioxidant potentials in fresh Polish, Ukrainian, and Israeli garlic samples after subjection to cooking temperature. Dietary fiber and essential trace elements were comparable. The antioxidant potentials were determined by four scavenging methods using beta-carotene, 1,1-diphenyl-2-picrylhydrazyl (DPPH), nitric oxide (NO), and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS(*)(+)) radical cation with K(2)S(2)O(8) or MnO(2) assays. Polyphenols, tocopherols, proteins, and antioxidant potentials were higher in Polish garlic, but not significantly (P > 0.05). The SDS- and native-PAGE electrophoretic patterns of all three fresh garlic samples were without significant differences. Most of the proteins were in the molecular mass range of 24-97 kDa, and the more intensive major bands were concentrated at 50 and 12 kDa. The 50 kDa protein nearly disappears and the intensity of the 12 kDa lectin bands slightly decreases during cooking. It was observed that the bioactive compounds, antioxidant potential, and proteins in garlic decrease significantly after 20 min of cooking at 100 degrees C (P < 0.05). In conclusion, (a) the bioactive compounds, electrophoretic patterns, and antioxidant potential of fresh Polish, Ukrainian, and Israeli garlic samples are comparable; (b) garlic samples subjected to 100 degrees C during 20 min preserve their bioactive compounds, antioxidant potential, and protein profile and are comparable with fresh garlic; and (c) fresh garlic should be added to dishes cooked at 100 degrees C in the last 20 min of the cooking process.

Cortical reorganization associated lower extremity motor recovery as evidenced by functional MRI and diffusion tensor tractography in a stroke patient

Purpose: Recovery mechanisms supporting upper extremity motor recovery following stroke are well established, but cortical mechanism associated with lower extremity motor recovery is unknown. The aim of this study was to assess cortical reorganization associated with lower extremity motor recovery in a hemiparetic patient. Methods: Six control subjects and a 17 year-old woman with left intracerebral hemorrhage due to an arterio-venous malformation rupture were evaluated. The motor function of the paretic (left) hip and knee had recovered slowly to the extent of her being able to overcome gravity for 10 months after the onset of stroke. However, her paretic upper extremity showed no significant motor recovery. Blood oxygenation level dependent (BOLD) functional MRI at 1.5 Tesla was used to determine the acutual location of cortical activation in the predefined regions of interest. Concurrently, Diffusion Tensor Imaging (DTI) in combination with a novel 3D-fiber reconstruction algorithm was utilized to investigate the pattern of the corticospinal pathway connectivity between the areas of the motor stream. All subjects' body parts were secured in the scanner and performed a sequential knee flexion-extension with a predetermined angle of 0–60° at 0.5 Hz. Results: Controls showed anticipated activation in the contralateral sensorimotor cortex (SM1) and the descending corticospinal fibers stemming from motor cortex. In contrast to control normal subjects, the stroke patient showed fMRI activation only in the unaffected (right) primary SM1 during either paretic or nonparetic knee movements. DTT fiber tracing data showed that the corticospinal tract fibers were found only in the unaffected hemisphere but not in the affected hemisphere.

Effect of dehulling of rapeseed on feed value and nutrient digestibility of rape products in pigs

In the presented study the influence of dehulling rapeseed on the composition of rapeseed meal (RM) and rapeseed cake (RC) and on its feed value for piglets and growing-finishing pigs was investigated. Before withdrawal of oil, rapeseed (variety Express) was dehulled applying a procedure developed by SKET GmbH Magdeburg and the Section Food-Technology of the University Essen. The steps of the dehulling procedure were described. For RM the oil was removed by the prepress-solvent procedure till a crude fat content of 2.1% in DM. RC was produced by pressing only resulting approximately 13% crude fat in DM. The RM and RC from not dehulled (ND) and dehulled (D) rapeseed were examined analytically. Crude nutrients, sugar and fibre substances, amino acids, some minerals and trace elements, fatty acids, glucosinolates and sinapine, and phytate were determined. By dehulling the seed the crude fibre content was decreased in RM and RC by approximately 40%. The ADF content declined by 35 and 39%, and the NDF content by 28% and 40% in RM and RC, respectively. The decrease in ADL content amounted to 50% and 65% for RM and RC, respectively. On the other hand, the CP content of RM and RC was increased by 7% and 13%, respectively, by dehulling the seed while the amino acid content of rape protein increased only slightly. The contents of glucosinolates and sinapine were also increased by dehulling, while the contents of phytate and phytate P were decreased. In digestibility and balance experiments with piglets and intact hybrid breeds of growing-finishing pigs, the digestibility of organic matter and of crude nutrients and the contents of digestible energy and metabolizable energy were estimated. Furthermore, the precaecal digestibility of crude nutrients and amino acids was determined with fistulated mini-pigs. By dehulling the seeds the digestibility of organic matter from RM and RC was improved in piglets and adult pigs by approximately 10%, and the ME contents increased by 13 – 15%. The precaecal digestibility of the sum of amino acids was increased by approximately 3 and 6 units in RM and RC, respectively. The precaecal digestibility of lysine in RM and RC reached that of soybean oil meal from not dehulled beans.

Olfactory ensheathing cell grafts have minimal influence on regeneration at the dorsal root entry zone following rhizotomy.

The effectiveness of grafts of olfactory ensheathing cells (OECs) as a means of promoting functional reconnection of regenerating primary afferent fibers was investigated following dorsal root injury. Adult rats were subjected to dorsal root section and reanastomosis and at the same operation a suspension of purified OECs was injected at the dorsal root entry zone and/or into the sectioned dorsal root. Regeneration of dorsal root fibers was then assessed after a survival period ranging from 1 to 6 months. In 11 animals, electrophysiology was used to look for evidence of functional reconnection of regenerating dorsal root fibers. However, electrical stimulation of lesioned dorsal roots failed to evoke detectable cord dorsum or field potentials within the spinal cord of any of the animals examined, indicating that reconnection of regenerating fibers with spinal cord neurones had not occurred. In a further 11 rats, immunocytochemical labeling and biotin dextran tracing of afferent fibers in the lesioned roots was used to determine whether regenerating fibers were able to grow into the spinal cord in the presence of an OEC graft. Although a few afferent fibers could be seen to extend for a limited distance into the spinal cord, similar minimal in-growth was seen in control animals that had not been injected with OECs. We therefore conclude that OEC grafts are of little or no advantage in promoting the in-growth of regenerating afferent fibers at the dorsal root entry zone following rhizotomy.

The thalamic paraventricular nucleus relays information from the suprachiasmatic nucleus to the amygdala: a combined anterograde and retrograde tracing study in the rat at the light and electron microscopic levels.

The relationship between efferents of the hypothalamic suprachiasmatic nucleus (SCN) and neurons of the thalamic paraventricular nucleus (PVT) projecting to the amygdala was investigated in the rat using tract tracing in light and electron microscopy. Biotinylated dextran amine was used to label anterogradely SCN efferents. These fibers were found to reach the thalamic midline, terminating in PVT, through three pathways: anterodorsally through the preoptic region, dorsally through the periventricular hypothalamus, and through the contralateral medial hypothalamic and preoptic areas after crossing the midline in the optic chiasm. Preterminal and terminal-like elements labeled from the SCN were distributed throughout the rostrocaudal extent of PVT, with an anteroposterior gradient of density. Labeled terminal elements were densest in the dorsal portion of PVT beneath the ependymal lining and some of them entered the ependyma. Anterograde tracing of SCN fibers was combined with injections of retrograde tracers in the amygdala. Numerous retrogradely labeled cell bodies were seen throughout PVT, with a prevalence in its anterodorsal portion. Overlap was detected between puncta labeled from the SCN and retrogradely labeled neurons, especially in the anterodorsal sector of PVT, where numerous puncta were in close apposition to thalamo-amygdaloid cells. Electron microscopy revealed that boutons labeled from the SCN established synaptic contacts with dendritic profiles of PVT neurons labeled from the amygdala. The findings demonstrate that information processed in the biological clock is conveyed to the amygdala through PVT, indicating that this nucleus plays a role in the transfer of circadian timing information to the limbic system.

Optical fibre instrumentation for environmental monitoring applications

We report our research on the development of optical fibre trace gas sensors for environmental applications. We describe the operation of a 64-point fibre-optic methane sensor, which has been installed on a landfill site in Glasgow, UK, where methane is used for power generation as part of the current trend for renewable energy programmes. Although the environmental conditions are harsh, the sensor has performed satisfactorily, detecting methane in the range of ~50 ppm to 100% methane. Another area of our current research is the application of erbium-doped fibre lasers and amplifiers in gas spectroscopy. One system under investigation consists of an all-fibre cavity ring-down loop employing a fibre amplifier for the compensation of loop loss. We have been able to obtain ring-down times as long as 0.2 ms, corresponding to ~1100 pulses in the loop, producing an effective increase in a gas cell length from 5 cm to 55 m. The mode-locked operation of fibre lasers is also under investigation and, using dispersion effects, we demonstrate fine tuning of the wavelength which is important for absorption line scanning, with a typical tuning rate of ~0.014 nm kHz−1 at the third harmonic, closely matching the theoretical predictions. Techniques for extending fibre laser systems to form multi-point, multi-species gas sensors are explored.

Lack of Enhanced Spinal Regeneration in Nogo-Deficient Mice

The failure of regeneration of severed axons in the adult mammalian central nervous system is thought to be due partly to the presence of endogenous inhibitors of axon regeneration. The nogo gene encodes three proteins (Nogo-A, -B, and -C) that have been proposed to contribute to this inhibition. To determine whether deletion of nogo enhances regenerative ability, we generated two lines of mutant mice, one lacking Nogo-A and -B but not -C (Nogo-A/B mutant), and one deficient in all three isoforms (Nogo-A/B/C mutant). Although Nogo-A/B-deficient myelin has reduced inhibitory activity in a neurite outgrowth assay in vitro, tracing of corticospinal tract fibers after dorsal hemisection of the spinal cord did not reveal an obvious increase in regeneration or sprouting of these fibers in either mouse line, suggesting that elimination of Nogo alone is not sufficient to induce extensive axon regeneration.

Regenerative process of the facial nerve: rate of regeneration of fibers and their bifurcations

After the main trunk of the mouse facial nerve was injured by crushing, a fiber tracing method was used to quantify the facial motor neurons that extended regenerating nerve fibers to the specific site of the facial nerve branch. The total number of motor neurons retrogradely labeled with a fluorescent tracer, Fluoro-Gold (FG), were 0 on postsurgical days (PSDs) 1 and 2, 75±25 on PSD3, 264±21 on PSD4, 378±19 on PSD6, 428±19 on PSD8, 491±13 on PSD12 and 532±15 on PSD16. Assuming that the FG-positive neurons (535±11) of the control mice represent 100%, the FG-labeled neurons accounted for 0, 14, 49, 71, 80, 92 and 99% on the corresponding days. Two different fluorescent tracers were applied to the different facial nerve branches 16 days after facial nerve injuries. Double-labeled neurons were consistently found in the nerve-crushed facial nucleus (3.2%), and their number increased in the nerve-transected facial nucleus (12.2%). The present study indicates that the regenerating facial nerve consists of heterogeneous nerve fibers with varying growth rates and that excessive axonal branching occurs more frequently in the nerve-transected than in the nerve-crushed injuries.