Measurements and mapping of 282,420 nerve fibers in the S1-5 nerve roots.

OBJECT: The study aims to analyze nerve fiber types in the sacral nerve roots as a prerequisite for stimulation. METHODS: One-micrometer cross-sections of human ventral and dorsal S1-5 roots were stained with osmium and toluidine blue. The total fiber diameter and myelin sheath were measured in 282,420 nerve fibers. RESULTS: The analysis revealed the following 3 main nerve fiber types: Aalpha fibers (diameter 6-14 microm), Agamma fibers (diameter 2-4 microm), and B fibers (diameter < 2 microm). The B fibers were absent in S-1, present in some S-2 fascicles, and abundant from S-3 to S-5. The Aalpha fibers dominated the S-1 roots and most fascicles of S-2 roots. In the S3-5 roots, only a few Aalpha fibers were present. The relative occurrence of Agamma fibers increased from S-1 to S-5. In dorsal roots, Agamma fibers represented approximately 70% of all nerve fibers in every root and fascicle. CONCLUSIONS: The B fibers represented efferent parasympathetic fibers. These fibers were concentrated in certain areas of the nerve roots, not randomly distributed. The Aalpha fibers innervate lower-extremity muscles and sphincters. The inverse correlation of Aalpha and Agamma fibers in the ventral roots from S-1 to S-5 is surprising. In dorsal roots, Agamma fibers may conduct pain, touch, and temperature signals. Highly selective fiber stimulation specific for type, location, and direction may improve sacral nerve stimulation for a spastic bladder in paraplegic individuals.

Intradural microanatomy of the nerve roots S1-S5 at their origin from the conus medullaris.

OBJECT: The conus medullaris and the nerve roots from S-1 to S-5 regulate bladder function as well as movement and sensation of the lower extremities. This most caudal region of the spinal cord has not been studied in great detail anatomically despite its important regulatory role. The goal of this analysis is to characterize the normal intradural microanatomy of the sacral nerve roots at their origin from the conus medullaris. METHODS: The thecal sacs from 20 cadavers were fixated in formaldehyde and dissected under the operative microscope. RESULTS: More than 50 rootlets originated from the conus medullaris over a distance of < 3 cm. The rootlets were loosely organized into bundles by the arachnoid membrane with decreasing diameters. These diameters were 1.7 mm (ventral)/2.4 mm (dorsal) at S-1, and 0.17 mm (ventral)/0.4 mm (dorsal) at S-5. The roots were separated by neither the dentate ligament nor interradicular gaps. The number of rootlets decreased in the rostrocaudal direction with 2 ventral and 5 dorsal rootlets at S-1, but only 1 ventral (inconsistently found) and 2 dorsal rootlets at S-5. Typically, 1 nerve anastomosis was present between adjacent dorsal roots from S-1 to S-4. Nerve anastomoses between ventral roots or rootlets of the same root were less frequent. The dorsal segment of origin (linea radicularis) decreased in length from 7.2 mm at S-1 to 4.8 mm at S-5. CONCLUSIONS: The current study provides anatomical details and specific morphometric data of the intradural contents at the level of the conus medullaris. This information is valuable for intraoperative orientation, endoscopic navigation, and possible intradural nerve stimulation.

Effects of steroid hormones on the Zn, Cu and MTI/II levels in the mouse brain.

The effects of some steroid hormones (corticosterone, hydrocortisone, testosterone and estrone) on the Zn, Cu metabolism and metallothioneins levels in the mouse brain were studied. To administrate the hormones, aqueous suspensions and olive oil solutions injected subcutaneously were used alternatively. The quantification of metals and metallothioneins concentrations in brain homogenates revealed significant alterations of both metal ions and protein expression levels, yet the subcutaneous oil injection increased per se the tissue metallothionein expression and metal content. We have also defined by immunohistochemistry the area-specific distribution of metallothioneins isoforms-I/II and of glial fibrillar acid protein. Upon treatment, corpus callosum, mesencephalon, pons, hippocampus and cerebellum were found to be the areas that increase the protein expression levels, whereas all other brain areas were marginally affected or were unaffected in terms of immunopositive metallothionein reaction. The metallothionein-I/II expression was compared with the immunopositivity of glial fibrillar acid protein and the results are discussed within the framework of the physiological role of corticosteroids and the potential therapeutical importance of sexual hormones.

Long-term effects of St. John's wort and hypericin on monoamine levels in rat hypothalamus and hippocampus.

Hypericum perforatum L. (St. John's wort) is one of the leading psychotherapeutic phytomedicines and, because of this, great effort has been devoted to clarifying its mechanism of action. Chronic effects of St. John's wort and hypericin, one of its major active compounds, on regional brain amine metabolism have not been reported yet. We used a high-performance liquid chromatography system to examine the effects of short-term (2 weeks) and long-term (8 weeks) administration of imipramine, Hypericum extract or hypericin on regional levels of serotonin (5-HT), norepinephrine, dopamine and their metabolites in the rat brain. We focused our interest on the hypothalamus and hippocampus, as these brain regions are thought to be involved in antidepressant drug action. Imipramine (15 mg/kg, p.o.), Hypericum extract (500 mg/kg, p.o.), and hypericin (0.2 mg/kg, p.o.) given daily for 8 weeks significantly increased 5-HT levels in the hypothalamus (P<0.05). The 5-HT turnover was significantly lowered in both brain regions after 8 weeks of daily treatment with the Hypericum extract (both P<0.05). Consistent changes in catecholamine levels were only detected in hypothalamic tissues after long-term treatment. Comparable to imipramine, Hypericum extract as well as hypericin significantly decreased 3,4-dihydroxyphenylacetic acid and homovanillic acid levels in the hypothalamus (P<0.01). Our data clearly show that long-term, but not short-term administration of St. John's wort and its active constituent hypericin modify levels of neurotransmitters in brain regions involved in the pathophysiology of depression.

Effects of chronic treatment with sodium tetrachloroaurate(III) in mice and membrane models.

Gold is a nonessential element with a variety of applications in medicine. A few gold(I) compounds are used in the clinics for treatment of rheumatoid arthritis and of discoid lupus. Some novel gold(III) compounds are under evaluation as anticancer agents. It is known that gold compounds generally produce toxic effects on the kidneys and characteristic lesions in the brain. However, information concerning the neurotoxicity of gold derivatives in humans as well as in experimental toxicology is rather scarce. For this reason we tried to shed some further light on this aspect of gold neurotoxicity by chronic treatment of mice with sodium tetrachloroaurate(III) in order to observe possible biophysical and morphological alterations that may occur in the brain. Chronic gold treatment resulted in a markedly decreased expression of metallothioneins and of glial fibrillary acidic protein in astrocytes of different brain areas. To examine its effects on cell membranes, interactions of sodium tetrachloroaurate(III) with molecular models were also evaluated. The models consisted in bilayers built-up of classes of phospholipids located in the outer and inner monolayers of biological membranes. Structural perturbation of cell membrane models was observed only at concentrations 10(5) times higher than those detected in the brains of animals after three months' treatment. These results show that toxic effects on animal brain upon treatment with sodium tetrachloroaurate develop with difficulty and may be observed only at high doses.

Circulating endothelial progenitor cells in kidney transplant patients.

BACKGROUND: Kidney transplantation (RTx) leads to amelioration of endothelial function in patients with advanced renal failure. Endothelial progenitor cells (EPCs) may play a key role in this repair process. The aim of this study was to determine the impact of RTx and immunosuppressive therapy on the number of circulating EPCs. METHODS: We analyzed 52 RTx patients (58±13 years; 33 males, mean ± SD) and 16 age- and gender-matched subjects with normal kidney function (57±17; 10 males). RTx patients received a calcineurin inhibitor (CNI)-based (65%) or a CNI-free therapy (35%) and steroids. EPC number was determined by double positive staining for CD133/VEGFR2 and CD34/VEGFR2 by flow cytometry. Stromal cell-derived factor 1 alpha (SDF-1) levels were assessed by ELISA. Experimentally, to dissociate the impact of RTx from the impact of immunosuppressants, we used the 5/6 nephrectomy model. The animals were treated with a CNI-based or a CNI-free therapy, and EPCs (Sca+cKit+) and CD26+ cells were determined by flow cytometry. RESULTS: Compared to controls, circulating number of CD34+/VEGFR2+ and CD133+/VEGFR2+ EPCs increased in RTx patients. There were no correlations between EPC levels and statin, erythropoietin or use of renin angiotensin system blockers in our study. Indeed, multivariate analysis showed that SDF-1--a cytokine responsible for EPC mobilization--is independently associated with the EPC number. 5/6 rats presented decreased EPC counts in comparison to control animals. Immunosuppressive therapy was able to restore normal EPC values in 5/6 rats. These effects on EPC number were associated with reduced number of CD26+ cells, which might be related to consequent accumulation of SDF-1. CONCLUSIONS: We conclude that kidney transplantation and its associated use of immunosuppressive drugs increases the number of circulating EPCs via the manipulation of the CD26/SDF-1 axis. Increased EPC count may be associated to endothelial repair and function in these patients.

Optimized flow cytometric analysis of endothelial progenitor cells in peripheral blood.

Although flow cytometry is a rapid and convenient way to measure the number of circulating endothelial progenitor cells (EPC), there is no standard technique for preparation and measurement. The aim of this study was to present an optimized preparation method for EPC measurement which should serve as a standard to facilitate the comparison of the results in stem cell investigations by different research groups. We have looked for the preparation method which delivered the best immunostaining with the directly conjugated antibodies against VEGF R2, CD133, CD34, and CD45. In order to test the sensitivity of the method, we determined the number of EPC in the peripheral blood of volunteers by flow cytometry and by cell culture assay. Furthermore, we have evaluated the influence of different durations of conservation on the EPC cell count. The pre-treatment of blood samples with 0.2% formaldehyde for 30 minutes delivers the best immunostaining, and blood samples can be stored overnight at 4 degrees C without loss of counting rate for EPC. We found an excellent correlation (r = 0.98) between the flow cytometric measurement and the cell count of the cell culture method. The presented protocol for the flow cytometric measurement of EPC in the peripheral blood can be used as a diagnostic or prognostic tool; we propose this protocol as the standard for EPC quantification.

Zn and Cu alteration in connection with astrocyte metallothionein I/II overexpression in the mouse brain upon physical stress.

The distribution of metallothioneins I/II in the mouse brain and their specific area distribution upon physical stress were studied. To induce physical stress, groups of mice were subjected to total darkness for different periods (2 weeks, 1 month, and 2 months). The concentration of metallothioneins, evaluated by immunohistochemistry, as well as area-specific protein expression, were found in the following quantitative order: corpus striatum, cerebellum, mesencephalon, hippocampus with fornix, parts of thalamus, and pons. All other brain areas were marginally affected, or even unaffected, in terms of immunopositive metallothionein reaction. Metallothionein I/II expression was compared with the immunopositivity of glial fibrillary acidic protein (GFAP). It is noteworthy that metallothioneins and GFAP are expressed in different types of astrocytes.

Rearrangement of the retino-collicular projection after partial optic nerve crush in the adult rat.

The establishment of retino-collicular topography is a well-investigated model of axon pathfinding and it was believed that this topography is irreversibly fixed throughout life. We now report that, after partial crush of the adult rat optic nerve, the anterograde transport of intravitreally-injected tracers via axons of surviving retinal ganglion cells (RGC) in all retinal quadrants is confined to the rostro-medial part of the superior colliculus (SC). This indicates that the retino-collicular topography is rearranged after partial crush of the adult rat optic nerve. The reorganization starts in the injured optic nerve where surviving axonal fibres are demyelinized and bundled in the periphery of the optic nerve distal to the crush site. This is followed by a displacement of surviving axons to the medial part of the optic tract (OT) within 2 weeks. The infiltration of macrophages with the subsequent production of tumour necrosis factor-alpha at the lesion site is a prerequisite for the altered retino-collicular projection as blockade of tumour necrosis factor-alpha signalling with the neutralizing antibody Infliximab abolishes reorganization in the SC and lateralization of RGC axons in the optic nerve and OT. This suggests that optic nerve inflammation is necessary for a progressive bundling of surviving RGC axons, probably via clearance of cellular debris which, in turn, may lead to a redistribution of RGC axons to the medial OT and rostro-medial SC.