Automated characterization of nerve fibers labeled fluorescently: determination of size, class and spatial distribution.

Morphological classification of nerve fibers could help interpret the assessment of neural regeneration and the understanding of selectivity of nerve stimulation. Specific populations of myelinated nerve fibers can be investigated by retrograde tracing from a muscle followed by microscopic measurements of the labeled fibers at different anatomical levels. Gastrocnemius muscles of adult rats were injected with the retrograde tracer Fluoro-Gold. After a survival period of 3 days, cross-sections of spinal cords, ventral roots, sciatic, and tibial nerves were collected and imaged on a fluorescence microscope. Nerve fibers were classified using a variation-based criterion acting on the distribution of their equivalent diameters. The same criterion was used to classify the labeled axons using the size of the fluorescent marker. Measurements of the axons were paired to those of the entire fibers (axons+myelin sheaths) in order to establish the correspondence between so-established axonal and fiber classifications. It was found that nerve fibers in L6 ventral roots could be classified into four populations comprising two classes of Aalpha (denoted Aalpha1 and Aalpha2), Agamma, and an additional class of Agammaalpha fibers. Cut-off borders between Agamma and Agammaalpha fiber classes were estimated to be 5.00+/-0.09 microm (SEM); between Agammaalpha and Aalpha1 fiber classes to be 6.86+/-0.11 microm (SEM); and between Aalpha1 and Aalpha2 fiber classes to be 8.66+/-0.16 microm (SEM). Topographical maps of the nerve fibers that innervate the gastrocnemius muscles were constructed per fiber class for the spinal root L6. The major advantage of the presented approach consists of the combined indirect classification of nerve fiber types and the construction of topographical maps of so-identified fiber classes.

Effect of nerve graft porosity on the refractory period of regenerating nerve fibers.

OBJECT: In the present study the authors consider the influence of the porosity of synthetic nerve grafts on peripheral nerve regeneration. METHODS: Microporous (1-13 microm) and nonporous nerve grafts made of a copolymer of trimethylene carbonate and epsilon-caprolactone were tested in an animal model. Twelve weeks after surgery, nerve and muscle morphological and electrophysiological results of regenerated nerves that had grown through the synthetic nerve grafts were compared with autografted and untreated (control) sciatic nerves. Based on the observed changes in the number and diameter of the nerve fibers, the predicted values of the electrophysiological parameters were calculated. RESULTS: The values of the morphometric parameters of the peroneal nerves and the gastrocnemius and anterior tibial muscles were similar if not equal in the rats receiving synthetic nerve grafts. The refractory periods, however, were shorter in porous compared with nonporous grafted nerves, and thus were closer to control values. CONCLUSIONS: A shorter refractory period enables the axon to follow the firing frequency of the neuron more effectively and allows a more adequate target organ stimulation. Therefore, porous are preferred over nonporous nerve grafts.

Morphometric analysis of the fiber populations of the rat sciatic nerve, its spinal roots, and its major branches.

Correspondence between the nerve composition and the functional characteristics of its fiber populations is not always evident. To investigate such correspondence and to give a systematic picture of the morphology of the rat hind limb nerves, extensive morphometric study was performed on the sciatic nerve, its founding dorsal and ventral spinal roots, and its major branches. Nerve histology was examined in semithin sections via microscopic image analysis. Variation in the density of myelinated fibers, fiber interspace, and nerve cross-sectional area was studied in individual roots and nerves. In the dorsal roots, fiber numbers and cross-sectional areas were directly linearly proportional to the spinal root level number. Constituent fiber populations were identified using multicomponent lognormal models, and an optimal model for every nerve or root was selected by using an information theoretic approach. For the dorsal and ventral roots and the sciatic and peroneal nerves, optimal fiber population models consisted of three components, whereas, for the tibial and sural nerves, two components were optimal. Functional identities of the revealed fiber populations were established by using calculations of corresponding conduction velocities according to Arbuthnott et al. (J. Physiol. [1980] 308:125-157) and anatomical considerations. It is anticipated that morphological parameters established in this study would advance the development of neural prostheses in humans. The proximodistal correspondences among the fiber populations of different nerves were established by parametric statistical comparisons. The proposed approach provides a conceptual framework for understanding the comparative anatomy of the peripheral nerves and spinal roots and can be further applied in other species.

Motor cortex stimulation for Parkinson's disease: a modelling study.

Chronic motor cortex stimulation (MCS) is currently being investigated as a treatment method for Parkinson's disease (PD). Unfortunately, the underlying mechanisms of this treatment are unclear and there are many uncertainties regarding the most effective stimulation parameters and electrode configuration. In this paper, we present a MCS model with a 3D representation of several axonal populations. The model predicts that the activation of either the basket cell or pyramidal tract (PT) type axons is involved in the clinical effect of MCS. We propose stimulation protocols selectively targeting one of these two axon types. To selectively target the basket cell axons, our simulations suggest using either cathodal or bipolar stimulation with the electrode strip placed perpendicular rather than parallel to the gyrus. Furthermore, selectivity can be increased by using multiple cathodes. PT type axons can be selectively targeted with anodal stimulation using electrodes with large contact sizes. Placing the electrode epidurally is advisable over subdural placement. These selective protocols, when practically implemented, can be used to further test which axon type should be activated for clinically effective MCS and can subsequently be applied to optimize treatment. In conclusion, this paper increases insight into the neuronal population involved in the clinical effect of MCS on PD and proposes strategies to improve this therapy.

Type grouping in rat skeletal muscle after crush injury.

OBJECT: Accuracy of reinnervation is an important factor that determines outcome after nerve injury and repair. Type grouping--the clustering of muscle fibers of the same type after reinnervation--can be used to investigate the accuracy of reinnervation. In this study, the degree of type grouping after crush injury in rats was compared with the clustering of muscle fibers after autografting or single-lumen nerve grafting. METHODS: Twelve weeks after sciatic nerve crush injury in rats, clustering of Type I muscle fibers was analyzed in the target muscle with adenosine 5'-triphosphatase staining. In addition, the number of regenerated axons was determined in the nerve distal to the crush injury. Results were compared with that of the authors' previous study. RESULTS: Type grouping was more abundant after crush injury than after autograft or single-lumen nerve graft repair. CONCLUSIONS: Crush injury leads to more clustered innervation of muscle fibers, probably because the Schwann cell basal lamina tubes are not interrupted as they are in autograft or artificial nerve graft repair. This finding adds to understanding the processes playing a role in nerve regeneration.

Antigenic compartmentation of the cerebellar cortex in the chicken (Gallus domesticus).

The chick is a well-understood developmental model of cerebellar pattern formation,but we know much less about the patterning of the adult chicken cerebellum. Therefore an expression study of two Purkinje cell stripe antigens-zebrin II/aldolase C and phospholipase Cbeta4 (PLCbeta4)-has been carried out in the adult chicken (Gallus domesticus). The mammalian cerebellar cortex is built around transverse expression domains ("transverse zones"), each of which is further subdivided into parasagittally oriented stripes. The results from the adult chicken reveal a similar pattern. Five distinct transverse domains were identified. In the anterior lobe a uniformly zebrin II-immunopositive/PLCbeta4-immunonegative lingular zone (LZ; lobule I) and a striped anterior zone (AZ; lobules II-VIa) were distinguished. A central zone (CZ; approximately lobules VIa-VIIIa,b) and a posterior zone (PZ; approximately lobules VIIIa,b-IXc,d) were distinguished in the posterior lobe. Finally, the nodular zone (NZ; lobule X) is uniformly zebrin II-immunoreactive and is innervated by vestibular mossy fibers. Lobule IXc,d is considered as a transitional region between the PZ and the NZ, because the vestibular mossy fiber projection extends into these lobules and because they receive optokinetic mossy and climbing fiber input. It is proposed that the zebrin II-immunonegative P3- stripe corresponds to the lateral vermal B zone of the mammalian cerebellum and that the border between the avian homologs of the mammalian vermis and hemispheres is located immediately lateral to P3-. Thus, there seem to be transverse zones in chicken that are plausible homologs of those identified in mammals, together with an LZ that is characteristic of birds.

Electrophysiology and morphometry of the Aalpha- and Abeta-fiber populations in the normal and regenerating rat sciatic nerve.

We studied electrophysiological and morphological properties of the Aalpha- and Abeta-fibers in the regenerating sciatic nerve to establish whether these fiber types regenerate in numerical proportion and whether and how the electrophysiological properties of these fiber types are adjusted during regeneration. Compound action potentials were evoked from isolated sciatic nerves 12 weeks after autografting. Nerve fibers were gradually recruited either by increasing the stimulus voltage from subthreshold to supramaximal levels or by increasing the interval between two supramaximal stimuli to obtain the cumulative distribution of the extracellular firing thresholds and refractory periods, respectively. Thus, the mean conduction velocity (MCV), the maximal charge displaced during the compound action potential (Q(max)), the mean firing threshold (V(50)), and the mean refractory period (t(50)) were determined. The number of myelinated nerve fibers and their fiber diameter frequency distributions were determined in the peroneal nerve. Mathematical modeling applied to fiber recruitment and diameter distributions allowed discrimination of the Aalpha- and Abeta-fiber populations. In regenerating nerves, the number of Aalpha-fibers increased fourfold while the number of Abeta-fibers did not change. In regenerating Aalpha- and Abeta-fibers, the fiber diameter decreased and V(50) and t(50) increased. The regenerating Aalpha-fibers' contribution to Q(max) decreased considerably while that of the Abeta-fibers remained the same. Correlation of the electrophysiological data to the morphological data provided indications that the ion channel composition of both the Aalpha- and Abeta-fibers are altered during regeneration. This demonstrates that combining morphometric and electrophysiological analysis provides better insight in the changes that occur during regeneration.