Development of nNOS-positive preganglionic sympathetic neurons in the rat thoracic spinal cord.

To gain a better understanding of the neuroplasticity of sympathetic neurons during postnatal ontogenesis, the distribution of neuronal nitric oxide synthase (nNOS) immunoreactivity was studied in sympathetic preganglionic neurons (SPN) in the spinal cord (Th2 segment) of female Wistar rats at different ages (newborn, 10-, 20-, 30-day-old; 2-, 6-month-old; 3-year-old). In all age groups, the majority of nNOS-immunoreactive (IR) neurons was observed in the nucleus intermediolateralis thoracolumbalis pars principalis. In the first month, the proportion of nNOS-IR neurons decreased significantly from 92 ± 3.4% in newborn to 55 ± 4.6% in 1-month-old, while the number of choline acetyltransferase (ChAT)-IR neurons increased from 74 ± 4.2% to 99 ± 0.3% respectively. Decreasing nNOS expression in the first 10 days of life was also confirmed by western blot analysis. Some nNOS-IR SPN also colocalized calbindin (CB) and cocaine and amphetamine-regulated transcript (CART). The percentage of NOS(+)/CB(-) SPN increased from 23 ± 3.6% in 10-day-old to 36 ± 4.2% in 2-month-old rats. Meanwhile, the proportion of NOS(+)/CART(-) neurons decreased from 82 ± 4.7% in newborn to 53 ± 6.1% in 1-month-old rats. The information provided here will also serve as a basis for future studies investigating the mechanisms of autonomic neuron development.

Morphologic study of nerve root and types of needle used in transforaminal injections.

BACKGROUND: The bevel type and location of the distal orifice of the needle may have relevance for potential complications occurring during transforaminal epidural injection. METHODS: We examined by scanning electron microscopy the structural aspects of spinal nerve root cuffs of 3 human cadavers, and 3 needle types used in transforaminal injections: 22-gauge Quincke spinal needles, 22-gauge blunt nerve block needles, and 20-gauge radiofrequency blunt needles. We made punctures in vitro in the spinal nerve root cuffs, and we studied the structures affected. RESULTS: There is fat tissue within the nerve root with irregular distribution. In needles with a round tip, the distal orifice maintained an extraneural location after puncture of the nerve root cuff. The length of the needle required to introduce the distal orifice completely inside the nerve root cuff was variable, depending on the type: shortest for a Quincke needle (1.8 mm), 4.1 mm for the Epimed, and longest for radiofrequency needles (5.7 mm). CONCLUSIONS: The layer of fat around nerve roots may prevent the contact of the needle tip with axons. The sharp needle tip entered the nerve root cuff more easily than the blunt tip in the cadaveric nerve root samples, measured in a qualitative manner. There is a need for clinical studies to ascertain if blunt needles may be safer than sharp needles for transforaminal injections.

Olfactory ensheathing cells, olfactory nerve fibroblasts and biomatrices to promote long-distance axon regrowth and functional recovery in the dorsally hemisected adult rat spinal cord.

Cellular transplantation, including olfactory ensheathing cells (OEC) and olfactory nerve fibroblasts (ONF), after experimental spinal cord injury in the rat has previously resulted in regrowth of severed corticospinal (CS) axons across small lesion gaps and partial functional recovery. In order to stimulate CS axon regrowth across large lesion gaps, we used a multifactorial transplantation strategy to create an OEC/ONF continuum in spinal cords with a 2-mm-long dorsal hemisection lesion gap. This strategy involved the use of aligned OEC/ONF-poly(D,L)-lactide biomatrix bridges within the lesion gap and OEC/ONF injections at 1 mm rostral and caudal to the lesion gap. In order to test the effects of this complete strategy, control animals only received injections with culture medium rostral and caudal to the lesion gap. Anatomically, our multifactorial intervention resulted in an enhanced presence of injured CS axons directly rostral to the lesion gap (65.0 +/- 12.8% in transplanted animals versus 13.1 +/- 3.9% in control animals). No regrowth of these axons was observed through the lesion site, which may be related to a lack of OEC/ONF survival on the biomatrices. Furthermore, a 10-fold increase of neurofilament-positive axon ingrowth into the lesion site as compared to untreated control animals was observed. With the use of quantitative gait analysis, a modest recovery in stride length and swing speed of the hind limbs was observed. Although multifactorial strategies may be needed to stimulate repair of large spinal lesion gaps, we conclude that the combined use of OEC/ONF and poly(D,L)-lactide biomatrices is rather limited.

Analysis of the diffusion weighted MR microscopy data of excised spinal cord of a rat on the basis of the model of restricted diffusion.

Anisotropic diffusion in the excised rat spinal cord saturated with 0.9% saline was investigated using MR microimaging with b-values up to 8000 s/mm2 for different diffusion times. Non-exponential transversal diffusion decay found in white matter (WM) and gray matter (GM) was fitted with 2 components (the "fast" and the "slow"). Significantly smaller non-exponential dependence was found for the longitudinal diffusion in the WM. Obtained results corresponding to restricted diffusion in the range from approximately 2 to approximately 7 microm were correlated with axon diameter distribution in the WM obtained from transmission electron micrographs. It was concluded that observed diffusion anisotropy in the spinal cord might be entirely explained by presence of the slow transversal component, arising from the restricted diffusion. The strict analytical description of the diffusion decay in nervous tissue requires taking into account continuous distribution of the space-scale of the restricting barriers. The simplified two-component analysis may be applicable for visualization of the nervous tissue in clinical practice.

[Attachment points of the posterior longitudinal ligament and their importance for thoracic and lumbar spine fractures]. / Anheftungspunkte des Lig. longitudinale posterius und deren Bedeutung für Wirbelkörperfrakturen.

Spine fractures with damage of the posterior wall of the vertebra often can be anatomically reconstructed by indirect reduction. Whether the posterior longitudinal ligament (PLL) is responsible for the reduction is still subject to debate. The aim of our investigation was to ascertain the role of the PLL in closed reduction of spine fractures by identifying the bony attachment points of this ligament. We performed a gross anatomical dissection, a light- and polarized microscopic investigation on 22 human cadaverous thoracic and lumbar spines to determine the points of attachment of the PLL. We found two layers of the PLL. The superficial layer runs from the first thoracic down to the third lumbar vertebra with a width of 0.4-1.0 cm and from there descends as a thin rudiment to the sacrum. The deep layer shows a segmental rhomboid structure. Lateral fibers are attached to the annulus fibrosus and at the rim of the adjacent vertebrae. Medial fibers are attached additionally to the posterior wall of the vertebral bodies by bridging the foramina basivertebralia. Since these foramina become enlarged in the caudal parts of the vertebral column, the number of attachment points at the posterior wall of the vertebral bodies decreases caudally. Good results for reconstruction of the posterior wall in vertebral fractures of the thoracic and upper lumbar spine can be explained by the anatomical situation of the PLL and stress the important role of the PLL in indirect reduction of spine fractures.

Determination of bone volume by osteocyte population.

During development and growth, biological tissues and organisms can control their size and mass by regulating cell number (Raff, 1992; Conlon and Raff, 1999). Later in life both cell number and organ mass decrease (Buetow, 1985). We demonstrate that the number density of bone cells buried in the calcified matrix (osteocyte lacunar density) predicts extracellular matrix volume for both cancellous and cortical bone in a broad cross-section of the population (males and females, age range 23-91 years, r(2) = 0.98). Our hypothesis is that bone mass is determined by the control of osteocyte number, and that this is a particular instance of the control of organ size through the social controls on cell survival and death (Raff, 1992; Conlon and Raff, 1999).

Structural features and thickness of the vertebral cortex in the thoracolumbar spine.

STUDY DESIGN: The thickness and structure of the vertebral body cortex were examined from sections of human cadaveric vertebrae. OBJECTIVES: The objectives were to identify the principal structural features of the cortex, to directly measure the minimum and maximum thicknesses of the cortex in the thoracolumbar spine, and to compare regional variations in the structure of the cortex. SUMMARY OF BACKGROUND DATA: The thickness of the vertebral cortical shell contributes to the compressive strength of the vertebral body. There is little consensus concerning the thickness and morphology of vertebral shell and endplate along the spine in existing data. METHODS: Human T1, T5, T9, L1, and L5 vertebral bodies (mean age 70.4 years) from 20 cadaveric spines were sectioned and photographed. The minimum and maximum cortical thickness of the shells and endplates in the midsagittal plane were measured from magnified images. RESULTS: The anterior shell thickness was significantly greater than the posterior shell and both endplates. Endplate thickness was greatest in the lower lumbar vertebrae. There was a significant decrease in cortex thickness over the central portion of endplates and shells, with a mean minimum thickness of 0.40 mm and a mean maximum thickness of 0.86 mm, with an overall mean of 0.64 +/- 0.41 mm. Increased porosity was also observed along the central regions of the cortical shells. In the lower thoracic and lumbar spine, a double-layered endplate structure was observed. CONCLUSIONS: Invasive techniques provide the only means to directly resolve the thickness and distribution of bone in the vertebral cortex. The cortex thickness and structure varies along the endplates and the anterior and posterior surfaces of the vertebral body. The implications of the so called double-layered endplate structure are unknown, but indicate the need for further study.

Spaceflight inhibits bone formation independent of corticosteroid status in growing rats.

Bone formation and structure have been shown repeatedly to be altered after spaceflight. However, it is not known whether these changes are related to a stress-related altered status of the corticosteroid axis. We investigated the role of corticosteroids on spaceflight-induced effects in rat pelvis and thoracic vertebrae. Thirty-six male Sprague-Dawley rats were assigned to a flight, flight control, or vivarium group (n = 12/group). Bilateral adrenalectomy was performed in six rats per group, the additional six rats undergoing sham surgery. Adrenalectomized (ADX) rats were implanted with corticosteroid pellets. On recovery from spaceflight, thoracic vertebrae and the whole pelvis were removed and processed for biochemistry, histomorphometry, or bone cell culture studies. The 17-day spaceflight resulted in decreased bone volume (BV) in the cotyle area of pelvic bones (-12%; p < 0.05) associated with approximately 50% inhibition of bone formation in the cancellous area of pelvic metaphyses and in thoracic vertebral bodies. The latter effect was associated with a decreased number of endosteal bone cells isolated from the bone surface (BS) in these samples (-42%; p < 0.05). This also was associated with a decreased number of alkaline phosphatase positive (ALP+) endosteal bone cells at 2 days and 4 days of culture, indicating decreased osteoblast precursor cell recruitment. Maintaining basal serum corticosterone levels in flight-ADX rats did not counteract the impaired bone formation in vertebral or pelvic bones. Moreover, the decreased ex vivo number of total and ALP+ endosteal bone cells induced by spaceflight occurred independent of endogenous corticosteroid hormone levels. These results indicate that the microgravity-induced inhibition of bone formation and resulting decreased trabecular bone mass in specific areas of weight-bearing skeleton in growing rats occur independently of endogenous glucocorticoid secretion.

Unbiased determination of cytokine localization in bone: colocalization of interleukin-6 with osteoblasts in serial sections from monkey vertebrae.

Few data are available describing the in vivo localization of cytokines in bone. The objective of this study was to describe the histological localization of interleukin-6 (IL-6) relative to osteoblasts (alkaline phosphate [ALP]-positive cells) and osteoclasts (tartrate-resistant acid phosphate [TRAP]-positive cells) in midsagittal, paraffin-embedded serial sections of thoracic 13 (T-13) vertebrae from 49 female cynomolgus monkeys. Serial sections 1 and 4 were immunostained for IL-6, section 2 was histochemically stained for TRAP, and section 3 was immunostained for ALP. Sixteen centrally located fields were measured in the cancellous compartment and grid alignment among sections was verified using image analysis. Using a Merz grid, IL-6 localized to 6% of the bone surface on sections 1 and 4, whereas TRAP localized to 8.5% and ALP to 12% of the bone surface. Colocalization was defined as positive staining within an 80 x 80 microm block in the first serial section that "overlapped" staining in either the corresponding block or its eight surrounding blocks within the second serial section. For each section, 1600 blocks were analyzed. Using Monte Carlo simulations, random colocalization was calculated to determine the statistical significance of experimental colocalizations. Colocalization of approximately 90% between the two IL-6 sections verified staining reproducibility and proper grid alignment among sections. Colocalization of TRAP and ALP was not statistically different from random (p 0.3). As identified using ALP- or TRAP-positive surfaces, there was significant IL-6 colocalization with osteoblasts (p < 0.003), but not with osteoclasts (p 0.3). These in vivo colocalization data support the hypothesis that osteoblasts produce and respond to IL-6.

Cytokinetic pattern in the thoracic spinal cord of chick embryos (incubation day 5-13) using PCNA staining and TUNEL method.

For the cytokinetic studies using spinal cords of chick embryos, chronological patterns of cell proliferation and programmed cell death (apoptosis) should be known. Information in the early stages of chick embryos is available while data on later stages are seldom available. To investigate the chronological patterns of cell proliferation and apoptosis in the thoracic spinal cord of normal chick embryos on incubation day 5, 6, 8, 10 and 13 (Hamburger and Hamilton stage 26-40), proliferating cell nuclear antigen (PCNA) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method were used. Cell proliferation was active at the germinal layer on days 5 and 6. It markedly declined on day 8 and became negligible on day 13. TUNEL-positive cells were mainly found in the germinal layer, the ventrolateral part of the mantle layer and the dorsal root ganglion. Compared to PCNA-positive cells, TUNEL-positive cells were sparse, especially after day 10, when only a few positive cells were scattered. These results will be used as a control data for the studies such as an experimental research for neural tube defects.

Effects of spaceflight and recovery on rat humeri and vertebrae: histological and cell culture studies.

Skeletal changes associated with spaceflight in the rat have been well documented, but few data are available on bone tissue and bone cell metabolism after subsequent on-Earth recovery. We therefore investigated the effects of microgravity and subsequent recovery on trabecular bone morphology and cellular activities in rat humeri and thoracic vertebrae and compared histomorphometric parameters in caudal vertebrae with the behavior of vertebral osteoblastic cells in culture. We report here that humeral weight showed normal growth during the experiment but was unaffected by spaceflight or recovery from spaceflight. However, the 14-day spaceflight resulted in inhibition of static indexes of bone formation in humeral proximal metaphyses and thoracic vertebral bodies. This was associated with a decrease in bone volume in humeral metaphyses. After 14 days of on-Earth recovery, osteoblastic and osteoid surfaces returned toward normal and bone volume was normalized in humeri, whereas the static bone formation parameters were not restored in thoracic vertebrae. In addition, histological indexes of bone formation and osteoblastic cell growth in vitro were not affected by spaceflight in caudal vertebrae. This study shows that rat humeri and thoracic and caudal vertebrae exhibit different patterns of response to spaceflight and subsequent on-Earth recovery, which could be due, at least in part, to the different loading pattern of these bones, and also to differences in bone turnover rate.

The role of cartilage in osteogenesis in the human fetal vertebral body.

The cartilage canals of the thoracic and lumbar vertebrae of fetuses ranging in crown-rump (C.R.) length from 33 to 180 mm were examined under light microscopy. The activity of the cells within these canals contributed to the anterolateral growth of the vertebral body. Bone formation was seen to commence in the center of the vertebral body. The cells of the connective tissue contained within the cartilage canals appeared to participate in the process of osteogenesis in multiple, discrete foci. In five-month-old fetuses, periosteal bone was noted on the anterior and posterior surfaces of the vertebral body, and calcification in the walls of the cartilage canals gave the histologic appearance of bone spicules.

Thoracic sympathetic trunk compression by osteophytes associated with arthritis of the costovertebral joint. Anatomical and clinical considerations.

Hyperostosis (lipping) due to costovertebral arthritis was found frequently (84.3%) impinging on the sympathetic trunks (ganglia and cord), rami communicantes and roots of the splanchnic nerves on both sides of the spine in more than 1,000 dissecting room cadavers examined and 34 cadavers of adult and elderly people specially dissected. As a result of the compression, the affected sympathetic structures were angulated, deflected from their course, enlarged and often infiltrated with connective tissue. The possible symptoms which may result from this kind of compression are discussed.

Microscopic organization of the ligamenta flava in Cercopithecinae.

The ligamenta flava have been investigated in Cercopithecus pygerythrus (vervet monkey) and C. mitis (blue monkey) at various levels of the vertebral column. It has been shown that in the cervical, thoracic and lumbar regions the topography of these ligaments differs. The cervical ligamenta flava do not extend over the intervertebral joints, while in the thoracic and lumbar regions they reinforce and fuse with the capsule. The ligamenta flava from T4 to S1 replace the interspinous ligaments by extending into the interspinous space. The ligamenta flava consist mostly of interwoven layers of elastic and collagen fibres. The area of attachment has four zones whose histological details are described. These findings are discussed in relation to the function of the vertebrbility of the column and mechanical adaptation to absorb sudden stresses.

Creep characteristics of the human spinal column.

Thirty-two static compression tests were carried out on 52 intervertebral disks and their posterior articular facet joints. The spinal units were tested within a uniquely designed test apparatus. Following the experiment, each intervertebral disk was examined macroscopically to assess the degree of disk degeneration. Three experiments were carried out at suitably chosen load levels, which were statically appropriate for a given vertebral level and its loading history. In the first experiment a constant load was applied to a spinal segment and its decrease in height was measured as a function of time. The results were as follows: Under the application of a constant load, the vertebral unit suffers a gradual change in its dimensions. This typical behavioral response pattern is identified as creep. Creep may be defined as the time dependent part of the deformation that accompanies the application of a load to the disk. The measured creep did not continue indefinitely but decreased with time until the vertebral specimen was equilibrated. The time to equilibration was a function of disk grade. The rate of creep was found to be dependent upon the condition of the nucleus pulposus. Creep is of considerable importance because of its influence on the kinetics and kinematics of the vertebral unit. The effect of creep is to increase the modulus of elasticity with time; hence a reduction occurs in the compliance of the spinal unit. In the second experiment, incremental loads were applied to a spinal unit. The unit was allowed to equilibrate and an additional load was applied. Its purpose was to further delineate spinal unit mechanics in terms of constant load and creep behavior. It was concluded from these tests that the nucleus plays an important mechanical role in influencing the behavioral response of the vertebral unit to constant compressive load. As nuclear maturation occurs, the stiffness characteristics of the spinal unit were noted to increase as dis its deflection characteristics. Spinal unit geometry no doubt also plays a role in influencing these results. In the last experiment, load-deflection curves were determined for the vertebral unit. It was found that load-deflection curves are not uniquely defined by the relationship between the variables of load and deflection. The time each value of load is maintained must also be taken into account. The physiological and biochemical factors responsible for the observed spinal unit behavior are outlined, and the importance of the articular facet joints in governing spinal unit deflection stressed. Although it has long been known that the intervertebral disk suffers a slow and continuous deformation when subjected to mechanical load, the concept of spinal unit deformation as a time dependent function of load is comparatively new.