Morphometry of human superficial dorsal and dorsolateral column fibres: significance to spinal cord stimulation.

In spinal cord stimulation (SCS) large diameter cutaneous (Abeta) fibres in the dorsal columns (DCs) are activated and have an inhibiting effect on the transmission of pain signals by Adelta and C fibres from the corresponding dermatome(s). The largest Abeta fibres can be activated up to a maximum depth of about 0.25 mm in the DCs. No data are available on the distribution of the large fibres in this superficial human DC layer at the common SCS levels Th(10-11). Such data are indispensable to improve the predictive capability of a computer model of SCS. The whole myelinated fibre population in the superficial 300 microm of the dorsal column (DC(0-300)) at Th(10-11 )of two human subjects was morphometrically analysed. Some data was obtained from a third subject. The superficial dorsolateral column (DLC(0-300)) was included in this analysis because it was hypothesized that large dorsal spinocerebellar tract fibres could also be activated by SCS. Only very few fibres larger than 10.7 microm were found: a mean of 68 (0.5%) in DC(0-300) and 114 (2%) in DLC(0-300). Considering that the effect of SCS is primarily attributed to activation of these largest fibres, it is concluded that a surprisingly small average amount of 2.4 fibres per running 0.1 mm width and 6 fibres per segmental division of the DC is involved. Distinct mediolateral heterogeneity in fibre composition was found in both DC(0-300) and DLC(0-300). In the DC(0-300), the mean diameter of fibres > or =7.1 microm increases significantly by 5% from medial to lateral. Density (i.e. number of fibres per 1000 microm(2)) and frequency (i.e. percentage of a fibre size group compared to its parent population) of the large fibres increase significantly from medial to lateral in the DC(0-300). For fibres > or =10.7 microm, these parameters increase by 200 and 269%, respectively. It is concluded that the difference in stimulation threshold of large Abeta fibres in the median and lateral DC can be mainly attributed to the absence and presence, respectively, of collaterals at the stimulation site. Marked differences were found between DC(0-300) and DLC(0-300). The largest DLC(0-300) fibres (> or =10.7 microm) have a 320% higher frequency and a 473% higher density. Their mean diameter is, however, only 2% larger. The largest DLC(0-300) fibres are not likely to be recruited by SCS, since they are not larger than their DC(0-300) counterparts, they lack collaterals (which would reduce the threshold stimulus substantially) and they are more remote from the stimulation electrode.

Preservation and staining of myelinated nerve fibers.

Six procedures are given for preservation of myelinated nerve fibers for light or electron microscopic studies. These procedures fall into two main categories: those with and those without aldehyde fixation. Essentially different effects are attained by application of tannic acid, saline, microwave or conventional heating, or a decreased temperature. All procedures end in osmication. Three main aspects of myelinated fiber morphology are taken into account when judging the quality of their preservation: axon, myelin sheath, and axon/myelin coherence. Each aspect can be preserved excellently, but always in combination with a less superior quality of the other two aspects. Superior myelin quality is attained using microwave irradiation, either with aldehydes to which tannic acid is added or without aldehyde fixatives. Superior axon quality is attained with aldehydes and (conventional) heating. Axon/myelin coherence is best preserved by decreasing the temperature during the rinse with saline. Another two procedures provide good, though less superior, preservation of both axon and axon/myelin coherence. Next, the fixed tissue is embedded in plastic blocks from which semithin and ultrathin plastic sections are cut for light and electron microscopy, respectively. In addition to the standard procedure for toluidine blue staining on semithin sections, two microwave-supported procedures are described, which can be used as alternatives if the staining result is unsatisfactory. Furthermore, a toluidine blue staining procedure is described for glycol methacrylate (GMA)-embedded material, which can be used if larger sections are needed.

White matter of the cerebellum of the chicken (Gallus domesticus): a quantitative light and electron microscopic analysis of myelinated fibers and fiber compartments.

Low magnification light microscopic examination of the white matter in appropriately stained avian and mammalian cerebellum reveals a mediolateral succession in which areas of large, heavily myelinated fibers alternate with areas containing nearly exclusively small fibers. A large fiber accumulation (LFA) and its medially adjoining small fiber area (SFA) form a fiber compartment, which, with related parts of cortex and central nuclei, constitutes a so-called cerebellar module. The composition and the apparent mediolateral heterogeneity of cerebellar fiber compartments was quantified in the chicken by morphometrical analysis of myelinated fiber profiles in light (LM) and electron (EM) microscopic micrographs. In LM versus EM, approximately 37% of the myelinated fiber population is neglected. This deficit concerns profiles that are smaller than 1.2 micron2 (diameter < 1.2 microns). EM analysis is therefore considered a prerequisite and forms the main part of this study. The myelinated fiber population has a left-skewed log normal size distribution. Ninety-nine percent of the myelinated fibers fall within the range of 0.1 to 20 microns2 (diameter = 0.4-5.0 microns) and 90% are even smaller than 7 micron2 (diameter < 3.0 microns). Small fibers are abundant in both parts of the compartment. Statistical comparisons provide quantitative confirmation of the LM distinction of LFAs and SFAs. It appears, moreover, that, apart from typical LFAs and SFAs, transitional zones rather than sharp borders can be distinguished between the two. The medial border of the LFA appears to be more sharply defined than its lateral border. Distinct mediolateral fluctuations were found with respect to fiber density (166-243 fibers/1,000 microns2), mean profile area (2.4-4.0 microns2), and interspace (31-47%). These differences reflect the contrast between LFA (lower density, larger mean profile area) and SFA (higher density, smaller mean profile area). The interspace discriminates less well between LFA and SFA but is often smaller in the LFA and larger in the SFA. The presented quantitative characteristics of mediolateral heterogeneity in the cerebellar fiber layer can be used as reference for morphometric studies on the different fiber systems of the cerebellar white matter and the functional organization of the compartments.

Preservation of myelinated fibers for electron microscopy: a qualitative comparison of aldehyde fixation, microwave stabilisation and other procedures all completed by osmication.

A qualitative comparison was made of a variety of electron microscopic preservation methods for nervous tissue, especially with respect to myelinated fiber areas. The methods studied were aldehyde perfusion/immersion fixation, aldehyde-tannic acid immersion fixation (stimulated by either microwave or conventional heating), microwave stabilisation, saline treatment with conventional heating (all with secondary osmication), and primary osmication. For all methods three morphological aspects, the ultrastructural quality of myelin sheath and axon and the coherence between the two were judged separately. It appears that the best version of each method studied is capable of providing a good overall ultrastructural result but always shows a preference for one or two of the three separate morphological aspects. When aiming at good axon quality together with good axon/myelin coherence, aldehyde perfusion/immersion, saline treatment or primary osmication are almost equivalent. Microwave stabilisation, on the other hand, can be chosen when good myelin quality has to be combined with good axon quality. For more specific purposes the following examples can be given. When excellent myelin quality is needed both microwave-stimulated aldehyde-tannic acid fixation or microwave stabilisation can be considered. When the preservation of the axon quality has priority the aldehyde-perfused tissue should be further immersed in a heated aldehyde-tannic acid solution. Primary osmication guarantees excellent axon/myelin coherence. Despite the differences in detail, a remarkable correspondence is stressed between the overall results of sometimes extremely different methods of tissue preservation. Probably they all guarantee a reliable reflection of the in vivo situation. With respect to the use of microwave irradiation for tissue preservation, it appeared that stabilisation procedures are rather capricious. However, if successful, the results are not inferior to those of aldehyde fixation.

Ultrastructural study on avulsion effects of the cat cervical moto-axonal pathways in the spinal cord.

After selective avulsion of the ventral root cervical 7 (C7) from the adult cat spinal cord, the intraspinal trajectories of the torn axons in the white matter were studied at different survival times. Two phases could be discerned: an early phase which showed changes that occurred up to 14 days after avulsion and a second phase from day 30 onwards. Two days postoperatively, considerably swollen, empty myelin sheaths occurred, which remained present up to 14 days after avulsion. A primary increase in the number of glial cells (microglia) was noted on days 2 and 4 after avulsion. Ultrastructurally, unmyelinated and myelinated terminal clubs were found 8 and 14 days after avulsion. These clubs were characterized as cones of growth, related to axonal regeneration. A second glial increase was present after 30 days. At that time, the entire moto-axonal pathway clearly showed a degeneration pattern. This finding was light microscopically confirmed by an increase of GFAP-positive astrocytes. During the first 30 days, a front of small calibre myelinated axons, starting at the transition zone of the grey and white matter traversed halfway through the moto-axonal pathway. However, on days 60 and 90 no further shift of the front had occurred.

Does microwave irradiation have other than thermal effects on histological staining of the mammalian CNS? A light microscopical study of microwave stimulated staining under isothermal conditions in man and rat.

The question whether or not microwave irradiation exerts other than thermal effects on histological staining is still a matter of controversy. The present study was undertaken to reveal or reject such a so far hypothetical non-thermal irradiation effect. A device was developed, which enables exposure of histological sections or tissue pieces to microwave irradiation under isothermal conditions, i.e. with synchronous removal of the internal heat produced. Three classical neuroanatomical staining methods were tested on human and rat CNS. As control, identical procedures were performed without simultaneous microwave irradiation. The experiments were performed at three different temperature levels ranging from 5 to 50 degrees C. In none of the cases studied was a light microscopically appreciable difference observed between the microwave and non-microwave versions of a stain at the same temperature. The hypothesis of a separate non-thermal effect of microwave irradiation on histological staining is therefore rejected.

The axonal response after ventral root avulsions at the cervical 7 level in the cat spinal cord.

The ventral cervical 7th root was avulsed from the adult cat spinal cord and the reaction of the torn axons in the underlying white matter was studied by light microscopy using histochemical staining for acetylcholinesterase and immunocytochemical staining for neurofilament and by electron microscopy, after different survival times. One day postoperatively some of the torn axonal ends were enlarged in the intraspinal trajectory of the root fibers in the white matter between the ventral horn and the pial surface as observed by acetylcholinesterase staining. The formation of terminal clubs had accordingly started at this postlesional survival time. From day 2, terminal clubs positive to neurofilament antibody were found. The number of terminal clubs increased during the first 4 days after the avulsion, remained present at 30 days and then disappeared gradually during the next months. One such unmyelinated axonal terminal club was studied at the ultrastructural level. From its distal end an unmyelinated protrusion emanated which appeared myelinated on the most distal part. This protrusion may be the ultrastructural parallel of the ramifications from terminal axonal clubs described by Ramon Y Cajal after avulsion.

The neurofilament architecture of the rat suprachiasmatic nucleus.

The neurofilament architecture within the suprachiasmatic nucleus of the rat was analyzed immunocytochemically using neurofilament monoclonal antibodies. The topographic distribution of neurofilament containing structures was restricted mainly to the ventral and caudal part of the suprachiasmatic nucleus, coinciding with the entrance area of the retino-suprachiasmatic fibres of this nucleus. Within the nucleus itself an axonal organization was present. The axons were grouped, forming clusters. These clusters existed of a core of myelinated axons surrounded by unmyelinated axons. The myelinated/unmyelinated axon ratio could reach 1:25. Within the nucleus the myelinated axons extended upwards to the middle part of the suprachiasmatic nucleus, where the fibers of the axon clusters fanned out.