The influence of physical activity during youth on structural and functional properties of the Achilles tendon.

Achilles tendinopathy is a highly prevalent sports injury. Animal studies show a growth response in tendons in response to loading in the immature phase but not after puberty maturation. The aim of this investigation was to examine the structural and material properties in long distance runners who were either physically active (HAY) or inactive (LAY) in young age. Twelve men in HAY group and eight men in LAY group participated. Structural, functional, and biochemical properties of Achilles tendon were estimated from magnetic resonance imaging, ultrasound video recordings, mechanical tests, and tendon biopsies, respectively. There was no difference between the groups with respect to tendon cross-sectional area or tendon free length. There was no difference between the groups with respect to maximal force or mechanical properties. The collagen content, enzymatic and nonenzymatic cross-link density did not differ between the groups, nor did collagen fibril density, diameter, and area. There was a correlation between age and pentosidine/collagen within the groups [(HAY: P < 0.05 and r(2) = 0.47) and (LAY: P < 0.05 and r(2) = 0.52)]. The data suggest that high or low activity during youth did not appreciably influence the mechanical, structural, or biochemical properties of the Achilles tendon in adult long distance runners.

Effects of LPS and dietary free fatty acids on MCP-1 in 3T3-L1 adipocytes and macrophages in vitro.

BACKGROUND: High levels of free fatty acids (FFA) have been suggested to be one of the underlying mechanisms for adipose tissue (AT) inflammation and dysfunction in obesity. Human AT produces several adipokines including monocyte chemoattractant protein-1 (MCP-1), which are involved in the pathogenesis of obesity-mediated inflammation. OBJECTIVE: In this study, we investigated the effects of lipopolysaccharide (LPS) and a panel of dietary FFA on MCP-1 gene and protein expression in adipocytes and macrophages. Furthermore, we investigated whether the effect of LPS and FFA were mediated through the toll-like receptor 4 (TLR4). METHODS: 3T3-L1 adipocytes and THP-1 macrophages were incubated for 24 h with the following FFA: monounsaturated fatty acid (oleic acid), saturated fatty acid (palmitic acid) and trans fatty acid (elaidic acid; 500 µM) with and without LPS (2 ng ml(-1)), and MCP-1 and TLR4 mRNA expression and MCP-1 protein secretion was determined. RESULTS: The results showed that LPS significantly increased MCP-1 and TLR4 expression and MCP-1 secretion in 3T3-L1 adipocytes, and that the MCP-1 expression was blocked by a TLR4 inhibitor (CLI095). The effects of the various FFA on MCP-1 mRNA expression and protein secretion in the adipocytes showed no significant changes either alone or in combination with LPS. In macrophages, palmitic acid increased MCP-1 mRNA expression by 1.8-fold (P<0.05), but oleic acid and elaidic acid had no effects. CONCLUSIONS: In conclusion, in 3T3-L1 adipocyte, the TLR4-agonist, LPS, stimulates the proinflammatory chemokine MCP-1. The different classes of FFA did not induce MCP-1 mRNA expression or protein secretion in the adipocytes, but the saturated FFA, palmitic acid, induced MCP-1 mRNA expression in macrophages, possibly because of the higher expression level of TLR4 in the macrophages than the adipocytes. Our results indicate that FFA may induce AT inflammation through proinflammatory stimulation of macrophages.

Chronic alterations in growth hormone/insulin-like growth factor-I signaling lead to changes in mouse tendon structure.

The growth hormone/insulin-like growth factor-I (GH/IGF-I) axis is an important stimulator of collagen synthesis in connective tissue, but the effect of chronically altered GH/IGF-I levels on connective tissue of the muscle-tendon unit is not known. We studied three groups of mice; 1) giant transgenic mice that expressed bovine GH (bGH) and had high circulating levels of GH and IGF-I, 2) dwarf mice with a disrupted GH receptor gene (GHR-/-) leading to GH resistance and low circulating IGF-I, and 3) a wild-type control group (CTRL). We measured the ultra-structure, collagen content and mRNA expression (targets: GAPDH, RPLP0, IGF-IEa, IGF-IR, COL1A1, COL3A1, TGF-ß1, TGF-ß2, TGF-ß3, versican, scleraxis, tenascin C, fibronectin, fibromodulin, decorin) in the Achilles tendon, and the mRNA expression was also measured in calf muscle (same targets as tendon plus IGF-IEb, IGF-IEc). We found that GHR-/- mice had significantly lower collagen fibril volume fraction in Achilles tendon, as well as decreased mRNA expression of IGF-I isoforms and collagen types I and III in muscle compared to CTRL. In contrast, the mRNA expression of IGF-I isoforms and collagens in bGH mice was generally high in both tendon and muscle compared to CTRL. Mean collagen fibril diameter was significantly decreased with both high and low GH/IGF-I signaling, but the GHR-/- mouse tendons were most severely affected with a total loss of the normal bimodal diameter distribution. In conclusion, chronic manipulation of the GH/IGF-I axis influenced both morphology and mRNA levels of selected genes in the muscle-tendon unit of mice. Whereas only moderate structural changes were observed with up-regulation of GH/IGF-I axis, disruption of the GH receptor had pronounced effects upon tendon ultra-structure.

Degeneration and regeneration of motor and sensory nerves: a stereological study of crush lesions in rat facial and mental nerves.

The aim of this study was to evaluate the degeneration and regeneration of a sensory nerve and a motor nerve at the histological level after a crush injury. Twenty-five female Wistar rats had their mental nerve and the buccal branch of their facial nerve compressed unilaterally against a glass rod for 30s. Specimens of the compressed nerves and the corresponding control nerves were dissected at 3, 7, and 19 days after surgery. Nerve cross-sections were stained with osmium tetroxide and toluidine blue and analysed using two-dimensional stereology. We found differences between the two nerves both in the normal anatomy and in the regenerative pattern. The mental nerve had a larger cross-sectional area including all tissue components. The mental nerve had a larger volume fraction of myelinated axons and a correspondingly smaller volume fraction of endoneurium. No differences were observed in the degenerative pattern; however, at day 19 the buccal branch had regenerated to the normal number of axons, whereas the mental nerve had only regained 50% of the normal number of axons. We conclude that the regenerative process is faster and/or more complete in the facial nerve (motor function) than it is in the mental nerve (somatosensory function).

The spatial rotator.

This paper presents a new local volume estimator, the spatial rotator, which is based on measurements on a virtual 3D probe, using computer assisted microscopy. The basic design of the probe builds upon the rotator principle which requires only a few manual intersection markings, thus making the spatial rotator fast to use. Since a 3D probe is involved, it is expected that the spatial rotator will be more efficient than the the nucleator and the planar rotator, which are based on measurements in a single plane. An extensive simulation study shows that the spatial rotator may be more efficient than the traditional local volume estimators. Furthermore, the spatial rotator can be seen as a further development of the Cavalieri estimator, which does not require randomization of sectioning or viewing direction. The tissue may thus be sectioned in any arbitrary direction, making it easy to identify the specific tissue region under study. In order to use the spatial rotator in practice, however, it is necessary to be able to identify intersection points between cell boundaries and test rays in a series of parallel focal planes, also at the peripheral parts of the cell boundaries. In cases where over- and underprojection phenomena are not negligible, they should therefore be corrected for if the spatial rotator is to be applied. If such a correction is not possible, it is needed to avoid these phenomena by using microscopy with increased resolution in the focal plane.

Quantitative assessment of macrophages in the muscularis externa of mouse intestines.

Quantification of intestinal cells is challenging for several reasons: The cell densities vary throughout the intestines and may be age dependent. Some cell types are ramified and/or can change shape and size. Additionally, immunolabeling is needed for the correct identification of cell type. Immunolabeling is dependent on both up- and down-regulation of the antigen being labeled as well as on the primary and secondary antibodies, the fixation, and the enhancement procedures. Here, we provide a detailed description of immunolabeling of CD169(+) cells and major histocompatibility class II antigen (MHCII(+) ) cells and the subsequent quantification of these cells using design-based stereology in the intestinal muscularis externa. We used young (5-weeks-old) and adult (10-weeks-old) mice. Cell densities were higher in jejunum-ileum, when compared with colon. In jejunum/ileum, the cell densities increased in oral-anal direction in adults, whereas the densities were highest in the midpart in young animals. In colon, the cell densities decreased in oral-anal direction in both groups of animals. Except for the density of MHCII(+) cells in colon, the cell densities were highest in young animals. Densities of CD169(+) and MHCII(+) cells did not differ, except in the colon of young animals where the CD169(+) density was almost twice as high as the MHCII(+) density. CD169 and MHCII antigens seem to be expressed simultaneously by the same cell in jejunum/ileum. We conclude that cell densities depend on both the age of the mouse and on the location in the intestines.

Igf1r+/CD34+ immature ICC are putative adult progenitor cells, identified ultrastructurally as fibroblast-like ICC in Ws/Ws rat colon.

The colon of Ws/Ws mutant rats shows impairment of pacemaker activity and altered inhibitory neurotransmission. The present study set out to find structural correlates to these findings to resolve mechanisms. In the colon of Ws/Ws rats, interstitial cells of Cajal associated with Auerbach's plexus (ICC-AP) were significantly decreased and ICC located at the submuscular plexus and intramuscular ICC were rarely observed based on immunohistochemistry and electron microscopy. Ultrastructural investigations revealed that there was no overall loss of all types of interstitial cells combined. Where loss of ICC was observed, a marked increase in fibroblast-like ICC (FL-ICC) was found at the level of AP. Immunoelectron microscopy proved FL-ICC to be c-Kit(-) but gap junction coupled to each other and to c-Kit(+) ICC; they were associated with enteric nerves and occupied space normally occupied by ICC in the wild-type rat colon, suggesting them to be immature ICC. In addition, a marked increase in immunoreactivity for insulin-like growth factor 1 receptor (Igf1r) occurred, co-localized with CD34 but not with c-Kit. A significantly higher number of Igf1r(+)/CD34(+) cells were found in Ws/Ws compared to wild-type rat colons. These CD34(+)/Igf1r(+) cells in the Ws/Ws colon occupied the same space as FL-ICC. Hence we propose that a subset of immature ICC (FL-ICC) consists of adult progenitor cells. Immunohistochemistry revealed a reduction of neurons positive for neuronal nitric oxide synthase. The functional capabilities of the immature ICC and the regenerative capabilities of the adult progenitor cells need further study. The morphological features described here show that the loss of pacemaker activity is not associated with failure to develop a network of interstitial cells around AP but a failure to develop this network into fully functional pacemaker cells. The reduction in nitrergic innervation associated with the Ws mutation may be the result of a reduction in nitrergic neurons.

The macrophage system in the intestinal muscularis externa during inflammation: an immunohistochemical and quantitative study of osteopetrotic mice.

Intestinal inflammation results in disturbed intestinal motility in humans as well as in animal models. This altered function of smooth muscle cells and/or the enteric nervous system may be caused by activation of macrophages in muscularis externa and a thereby following release of cytokines and chemokines that causes influx of mononuclear cells and neutrophilic granulocytes. We subjected osteopetrotic (op/op) mice that lack certain macrophage subtypes, e.g. macrophages in the muscularis externa and +/+ mice to LPS to induce inflammatory cell influx. The densities of F4/80+, MHCII+, and myeloperoxidase+ cells were quantified using stereological sampling. In +/+ mice we found that MHCII+ cells outnumber F4/80+ cells and that LPS injection increased the density of MHCII+ cells temporarily but not that of F4/80+ cells. This indicates that an upregulation of MHCII antigen takes place and that two or more macrophage subtypes with comparable morphologies exist. Osteopetrotic mice lacked MHCII+, CD169+, and F4/80+ cells after either treatment, which indicate that these cells are CSF-1-dependent. LPS induced VCAM-1 activation of the vessels, modest influx of granulocytes, as well as an iNOS-activation in a cell type different from macrophages in both +/+ and op/op mice.

Pacemaker activity and inhibitory neurotransmission in the colon of Ws/Ws mutant rats.

The aim of this study was to characterize the pacemaker activity and inhibitory neurotransmission in the colon of Ws/Ws mutant rats, which harbor a mutation in the c-kit gene that affects development of interstitial cells of Cajal (ICC). In Ws/Ws rats, the density of KIT-positive cells was markedly reduced. Wild-type, but not Ws/Ws, rats showed low- and high-frequency cyclic depolarization that were associated with highly regular myogenic motor patterns at the same frequencies. In Ws/Ws rats, irregular patterns of action potentials triggered irregular muscle contractions occurring within a bandwidth of 10-20 cycles/min. Spontaneous activity of nitrergic nerves caused sustained inhibition of muscle activity in both wild-type (+/+) and Ws/Ws rats. Electrical field stimulation of enteric nerves, after blockade of cholinergic and adrenergic activity, elicited inhibition of mechanical activity and biphasic inhibitory junction potentials both in wild-type and Ws/Ws rats. Apamin-sensitive, likely purinergic, inhibitory innervation was not affected by loss of ICC. Variable presence of nitrergic innervation likely reflects the presence of direct nitrergic innervation to smooth muscle cells as well as indirect innervation via ICC. In summary, loss of ICC markedly affects pacemaker and motor activities of the rat colon. Inhibitory innervation is largely maintained but nitrergic innervation is reduced possibly related to the loss of ICC-mediated relaxation.

Motility patterns and distribution of interstitial cells of Cajal and nitrergic neurons in the proximal, mid- and distal-colon of the rat.

The aim of this work was to study the patterns of spontaneous motility in the circular and longitudinal muscle strips and to characterize the distribution of c-kit positive interstitial cells of Cajal (ICCs) and nitrergic neurons (nNOS) in the proximal, mid- and distal-colon of Sprague-Dawley rats. We described two types of spontaneous contractions: high frequency (HF) and low frequency (LF) contractions, which were recorded in the presence of tetrodotoxin, suggesting a non-neurogenic origin. Regional differences were found in the motility patterns depending on the muscle layer and on the part of the colon studied. Muscle strips without submuscular plexus (SMP) showed only LF contractions. The density of ICCs was of the same magnitude along the extent of the colon: about 90-120 cells mm(-2) at Auerbach's plexus (AP) and 50-60 cells mm(-2) at the SMP. nNOS positive cells were found at the level of the AP and the major density was found in the mid-colon. Electrical field stimulation abolished LF but did not affect HF contractions. Our results indicate that HF contractions are due to the ICC network found associated with the submuscular plexus (ICC-SMP). The origin of LF contractions is still unknown.

Chronic lithium treatment with or without haloperidol fails to affect the morphology of the rat cerebellum.

We used unbiased stereological principles to determine whether long-term administration of lithium at human therapeutic levels, with or without haloperidol, affects the number or sizes of cerebellar Purkinje cells or the volume of histological layers in the rat cerebellum. Twenty-eight rats were randomly divided into three groups, receiving either no treatment, lithium, or lithium combined with haloperidol. The serum lithium levels ranged from 0.50 to 0.77 mmol/l. Haloperidol was given at a daily dose of 1 mg/kg. After 30 weeks of treatment, the animals were killed and the cerebelli were histologically prepared. No statistically significant differences were observed between the groups with respect to the cerebellar measures.

Collagen fibril size and crimp morphology in ruptured and intact Achilles tendons.

The present study examined the hypothesis that collagen fibril diameter and crimp angle in ruptured human Achilles tendons differed from that of intact ones. Tissue samples were obtained from the central core (distal core) and the posterior periphery (distal superficial) at the rupture site, and the proximally intact (proximal superficial) part of the tendon in 10 subjects (38+/-8 years) with a complete tendon rupture. For comparisons corresponding tissue samples were procured from age (38+/-7 years) and gender matched intact Achilles tendons during routine forensic autopsy. The cross-sectional area density and diameter distribution of fibrils were analyzed using stereological techniques of digitized electron microscopy biopsy cross-sections, while crimp angle was measured by the changing banding pattern of collagen fibers when rotated between crossed polars. Nine of 10 persons with tendon ruptures reported that the injury did not occur during exceedingly large forces, and none experienced any symptoms in the days or months prior to the injury. Fibril diameter distribution showed no region-specific differences in either the ruptured or intact tendons for either group. However, in the distal core there were fewer fibrils in the ruptured compared to the intact tendons in 60-150 nm range, P<0.01. Similarly, in the distal superficial portion there were fewer fibrils in the ruptured compared to the intact tendons in the 90-120 nm range, 2P<0.05, while there were no differences in the proximal superficial tendons. Crimp angle did not display any region-specific differences, or any difference between the rupture and intact tendons. In conclusion, these data suggest that although crimp morphology is unchanged there appears to be a site-specific loss of larger fibrils in the core and periphery of the Achilles tendon rupture site. Moreover, the lack of symptoms prior to the rupture suggests that clinical tendinopathy is not an etiological factor in complete tendon ruptures.

Spontaneous axonal regeneration in rodent spinal cord after ischemic injury.

Here we present evidence for spontaneous and long-lasting regeneration of CNS axons after spinal cord lesions in adult rats. The length of 200 kD neurofilament (NF)-immunolabeled axons was estimated after photochemically induced ischemic spinal cord lesions using a stereological tool. The total length of all NF-immunolabeled axons within the lesion cavities was increased 6- to 10-fold at 5, 10, and 15 wk post-lesion compared with 1 wk post-surgery. In ultrastructural studies we found the putatively regenerating axons within the lesion to be associated either with oligodendrocytes or Schwann cells, while other fibers were unmyelinated. Immunohistochemistry demonstrated that some of the regenerated fibers were tyrosine hydroxylase- or serotonin-immunoreactive, indicating a central origin. These findings suggest that there is a considerable amount of spontaneous regeneration after spinal cord lesions in rodents and that the fibers remain several months after injury. The findings of tyrosine hydroxylase- and serotonin-immunoreactivity in the axons suggest that descending central fibers contribute to this endogenous repair of ischemic spinal cord injury.

Stereological quantification of lymphocytes in skin biopsies from atopic dermatitis patients.

Atopic dermatitis (AD) is histologically characterized by lymphocytic infiltration of the skin and quantitative assessment is required. This study introduces stereological techniques to quantify the number of lymphocytes in skin biopsies. Four-millimetre punch biopsies were taken from skin with active eczema in 8 adults with AD and from clinically normal skin from 4 of the patients. Five persons without allergy or skin disease served as controls. The mean number of lymphocytes in 4-mm skin biopsies was 469,000 and 124,000 in active eczema and in clinically normal skin, respectively. Compared with controls, the number of lymphocytes in biopsies increased by a factor of 6.8 in active eczema and a factor of 1.8 in clinically normal skin. If 20% of skin is affected by eczema the total number of lymphocytes located in the affected skin can be estimated to 1.27 x 10(10). A patient with clinically moderate AD has a considerable number of lymphocytes in the skin.

Does long-term physical exercise counteract age-related Purkinje cell loss? A stereological study of rat cerebellum.

Physical exercise affects properties of the central nervous system that may increase the brain's ability to counteract degenerative changes. We have previously reported that rats trained from 5 to 23 months of age have less age-related decrease in spontaneous motor activity than sham-treated sedentary rats. Each rat ran at a speed of 20 m/min on a horizontal treadmill, for 20 minutes, two times per day, 5 days a week. In the present study we have carried out stereological analyses of the cerebella of the same rats. The total number of Purkinje cells was estimated with the optical fractionator technique, the local volumes of individual Purkinje cells with the planar rotator technique, and the volumes of the cerebellar layers with Cavalierìs principle. We found that sedentary aged rats have 11% fewer Purkinje cells and 9% smaller Purkinje cell soma volumes (both 2P = 0.02) than exercised aged rats, and that exercised aged rats have the same number of Purkinje cells as young rats. These findings indicate that the degree of age-associated degenerative changes in parts of the central nervous system is dependent on earlier life style and health habits and may be prevented or delayed by physical exercise.

Neuron loss in cerebellar cortex of rats exposed to mercury vapor: a stereological study.

Mercury vapor produces tremor in humans and experimental animals. We have previously reported that mercury vapor intoxication over an 8-week period induces only subtle changes in dorsal root ganglia and nerve roots in rats. In the present study we have carried out stereological analyses of the cerebellum of the same rats, and demonstrated significant losses of Purkinje cells (12.7%, 2P = 0.005) and granule cells (15.6%, 2P = 0.016). All sizes of Purkinje cells were lost with an equal probability, i.e. there were no indication of any preferential loss of any subpopulation of the neurons. The volume of the granular cell layer was significantly reduced (18.9%, 2P = 0.0 15), whereas the volumes of the molecular layer and the white matter were unchanged. Previous stereological studies have demonstrated that methyl mercury intoxication primarily induces degeneration in the peripheral nervous system, while sparing the cerebellum. We therefore suggest that metallic mercury vapor and methyl mercury have different toxicological profiles in rats, where metallic mercury vapor mainly affects the central nervous system and methyl mercury mainly affects the peripheral nervous system.

Quantitative study of neurofilament-positive fiber length in rat spinal cord lesions using isotropic virtual planes.

Spontaneous reocurrence of neurofilament (NF)-positive fibers has been described after spinal cord lesions in rats. However, previously introduced methods to evaluate the lesion and the regenerative fiber outgrowth suffer from several biases, why a new concept of quantitative, morphological analysis after spinal cord injury is needed. Length quantification of the putatively spontaneously regenerating fibers has been difficult until recently, when two length estimators based on sampling with isotropic virtual planes within thick physical sections were introduced. The applicability of these techniques to estimate the total length of NF-positive fibers was evaluated in photochemically induced ischemic lesions of thoracic spinal cords in young rats 6 weeks postlesion. Fiber length was found to be the most consistent measure with a mean of 3.71 m (coefficient of variation, CV = 0.16) in the 0.90 mm3 (CV = 0.26) large lesions. Whether or not the NF-positive fibers observed inside the lesion represent spontaneously regenerating axons needs to be confirmed in longitudinal, functional, and ultrastructural studies.

Degeneration and regeneration in rabbit peripheral nerve with long-term nerve cuff electrode implant: a stereological study of myelinated and unmyelinated axons.

Selective and dynamically co-ordinated functional electrical stimulation (FES) of paralysed/paretic limbs in upper motor neuron lesioned people depends on optimal contact at the neural interface. Implanted nerve cuff electrodes may form a stable electrical neural interface, but may also inflict nerve damage. In this study the immediate and long-term effects of cuff implantation on the number and sizes of myelinated and unmyelinated axons have been evaluated with unbiased stereological techniques. Cuff electrodes were implanted in rabbit tibial nerves just below the knee joint, and the stereological analyses were carried out 2 weeks and 16 months after implantation. Myelinated axons were analysed at standardised levels proximal to, underneath, and distal to the cuff; unmyelinated axons underneath the cuff. A 27% loss of myelinated axons was found underneath and distal to the nerve cuff 2 weeks post surgery. All axonal sizes were equally lost except for the very smallest. At 16 months post surgery the number of myelinated axons was restored to control values at both levels. Except for the presence of regenerative sprouts at 2 weeks post surgery, no changes in the number or sizes of unmyelinated axons were revealed at either 2 weeks or 16 months post surgery. Our study demonstrates that implanted cuff electrodes may cause an initial loss of myelinated axons but with subsequent regeneration.

A stereological study of dorsal root ganglion cells and nerve root fibers from rats exposed to mercury vapor.

Although mercury vapor is known to produce tremor and peripheral neuropathy, neuropathological studies of the effects of the vapor are few in number. The aim of the present study has been to evaluate the effect of mercury vapor on the morphology of the dorsal root ganglion and the spinal nerve roots. Adult male rats were exposed to mercury vapor for 33 days. The exposed rats developed somatic signs of intoxication and became increasingly irritable. The total numbers and volumes of A- and B-cell perikarya in the dorsal root ganglia, the total number of myelinated axons in the roots, and the cross-sectional areas of axon and myelin in the nerve roots were estimated using unbiased stereological principles. The mean cross-sectional area of myelin associated with nerve fibers in dorsal nerve roots of the exposed group was significantly reduced by 20% (2P=0.014). A tendency towards a reduction was seen in axon area of myelinated nerve fibers in the dorsal nerve roots (2P=0.087) and in the total numbers and mean volume of A-cell perikarya (2P = 0.059 and 2P=0.087, respectively). No differences between the two test groups were found for any of the parameters measured in B-cells and ventral nerve roots. It is concluded that mercury vapor, in a dose sufficient to produce intoxication, induces only minor changes in dorsal root ganglion and nerve roots in rats.