Motor and sensory reinnervation of fast and slow mammalian muscles

Summary 1. The formation of endplates outside the original endplate region of a muscle fibre was studied in slow and fast rat muscles. It was found that such new endplates are readily formed on the soleus muscle, whereas hardly at all in the fast extensor digitorum longus. Most new endplates appear to be morphologically normal within 2 months after nerve implantation.2. The time course of recovery of slow and fast cat muscles was followed after crushing the sciatic nerve. It was found that the slow soleus muscle recovers more rapidly than the fast flexor hallucis longus muscle.3. The endplates of reinnervated cat muscles are more complicated than those of the control muscles, but have nevertheless fewer nerve terminals per endplate. Reinnervated muscles are more sensitive to curare and it is suggested that this is due to a decrease in transmitter release, for it was found that less acetylcholine is released from reinnervated rat hemidiaphragms than from control ones.4. Motor and sensory reinnervation of spindles and tendon organs was studied. At the time when motor reinnervation is almost completed the sensory endings from spindles and tendon organs are highly abnormal. Thus sensory reinnervation proceeds much more slowly than motor.     

Adaptation of muscle fibre types and capillary network to acute denervation and shortlasting reinnervation

We postulated that, in rat extensor digitorum longus muscle (EDL), the length of capillaries per fibre surface area (Lcap/Sfib) and per fibre volume (Lcap/Vfib) could reflect fibre-type transformations accompanied by changes in oxidative metabolic profile and selective fibre-type atrophy. We excised rat EDL muscle 2 weeks after the sciatic nerve was cut (acute denervation; DEDL) and 4 weeks after the nerve was crushed (early reinnervation; REDL) and characterised muscle fibre-type transformation by the expression of myosin heavy-chain isoforms and by succinate dehydrogenase (SDH) and nicotinoamide adenine dinucleotide-tetrazolium reductase (NADH-TR) reactions. The numerical percentage (N/N) and area percentage (A/A) of pure and hybrid fibres and their diameter were determined, as was the A/A of SDH- and NADH-TR-positive fibres. The length of capillaries per fibre length (Lcap/Lfib), Lcap/Sfib and Lcap/Vfib were estimated in REDL and Lcap/Vfib in DEDL. In DEDL, the type 2x and 2b fibres evidently atrophied, with the N/N of type 2x fibres being lower and that of hybrid fibres higher. In REDL, the N/N of hybrid fibres was even higher, consequent to a lower N/N of type 2b fibres; however, fibre diameters approached values of the control EDL. Compared with control EDL, denervated and reinnervated muscles exhibited a higher A/A of oxidative fibres. This is probably the result of fibre-type transformation and selective fibre atrophy. We conclude that capillary length does not change during acute denervation and early reinnervation. The obtained higher values of Lcap/Sfib and Lcap/Vfib are related to changes in muscle fibre cross-sectional area. This study was supported by the Slovenian Research Agency and the Ministry of Education, Youth and Sport of the Czech Republic (KONTAKT grant no. 9-06-6 and grant no. LC06063).     

大鼠骨骼肌快肌肌钙蛋白T的同工型

骨骼肌快肌的收缩主要是由钙离子通过肌钙蛋白所调节控制。这些肌钙蛋白位于肌纤维之中。肌蛋白包括肌钙蛋白T、肌钙蛋白C、肌钙蛋白I。采用双向聚丙烯酰胺凝胶电泳和免疫学技术,对大鼠胚胎、新生大鼠和成年大鼠的骨能肌快肌肌钙蛋白T的同工型进行了研究。在成年大鼠的骨能肌快肌中,发现了10种肌钙蛋白T同工型。在大鼠胚胎和新生大鼠的骨能肌中,发现了7种肌钙蛋白T同工型。作为不同动物、不同发育阶段和不同组织发育的特殊标记,这些肌钙蛋白T同工型具有重要意义[动物学报49(3)：362—369,2003]。     

Early postnatal growth of skeletal muscle blood vessels of the rat

The development of blood vessels during the first three postnatal weeks was studied in the ventral stripe of the spinotrapezius muscle of the rat by use of India ink-gelatine injections, and electron microscopy. The number of terminal arterioles and collecting venules remained unchanged postnatally in the observed area. A remarkable proximodistal gradient of vascular development was apparent: while the basic structure of the hilar vessels remained unchanged in the time studied, the intramuscular arteries and veins matured gradually. More peripherally, gradual maturation of terminal and precapillary arterioles was observed. The capillary endothelium and the pericytes showed immature features, and remained unchanged during the time studied. An intense rebuilding activity was found in the endothelial cells of the growing venules, expressed by various forms of gaps, covered by an intact basal lamina and pericytes. Numerous mast cells and macrophages were found along all vessels. Intramuscular lymphatics were not present prior to the first postnatal week.     

Resident macrophages (ED2- and ED3-positive) do not phagocytose degenerating rat skeletal muscle fibres

Two soleus muscles with degenerating muscle fibres were serially sectioned and adjacent sections from various levels of the skeletal muscles were stained with antibodies that react with either monocytes and inflammatory macrophages (ED1) or with the major subpopulations of resident macrophages (ED2 and ED3). ED2⁺ and ED3⁺ resident macrophages were abundant throughout the muscles but were not present within the degenerating fibres. ED1⁺ cells, in contrast, were rarely observed within the undamaged regions of the muscles but were abundant within the degenerating fibres and in the perimysium between arterioles and degenerating fibres. It is concluded that the phagocytosis of damaged muscle fibres is not carried out by the major subpopulations of resident macrophages.     

Effect of denervation and tenotomy on slow and fast muscles of the chicken

Summary Changes of muscle weights, fiber diameters and ultrastructure were studied in the slow anterior latissimus dorsi (ALD) and in the fast posterior latissimus dorsi (PLD) of the chick three weeks after denervation and tenotomy, and after combined denervation and tenotomy of the two muscles.The slow ALD muscle becomes hypertrophic after denervation (Feng, Jung and Wu, 1962). Three weeks after nerve section, wet weights of ALD muscles are increased by 60% and fiber diameters become by 30% larger than those of contralateral control muscles. In spite of this hypertrophy, degenerative changes are seen in the ultrastructure, similar to those described in denervated atrophic muscles. Areas of dedifferentiation with autophagic vacuoles and aggregates of tubules are found in superficial layers of some fibers. Disintegration of Z lines and filaments along one or two sarcomeres occurs in a number of myofibrils, especially in muscles of young animals.In contrast to denervation alone, simultaneous denervation and tenotomy of the ALD muscles results in atrophy. Decrease of muscle weights and reduction of fiber diameters are similar as after tenotomy; in both cases muscle fibers waste by degeneration and atrophy of myofibrils.The fast PLD muscles underwent extensive atrophy in all three series of experiments. Corresponding atrophic and degenerative changes of ultrastructure were found in all instances.The authors wish to acknowledge gratefully the skillful technical assistance of Mrs. M. Sobotková and Ing. M. Doubek, and editorial assistance of Miss Virginia Hamilton.     

Surface area of apical and basolateral plasmalemma of epithelial cells of the ductuli efferentes testis of the rat

Serial sectioning was used to determine the occurrence of ciliated cells, and a morphological technique was used to estimate the relative and absolute surface areas of apical and basolateral membrane of the epithelial cells lining the ductuli efferentes of the rat. It was found that the ciliated cells constitute 15% of the epithelial cells and occur as groups of mainly 1–3 cells which are distributed at random in the duct epithelium. For the non-ciliated cells it was estimated that the formation of microvilli by the apical membrane increased the surface area of that border by a factor of 37-fold. The average surface density of the basolateral membrane was 76% the surface density of the apical membrane. However, there was a 3-fold increase in surface density along the apicalbasal axis of the basolateral plasmalemma. In the Discussion, the ductuli efferentes are compared to their homologue, the proximal tubules of the kidney, in the rates of fluid transport and membrane adaptations of their epithelium.     

Postnatal variations in the number and size of C-cells in the rat thyroid gland

The heterogeneous distribution of thyroid C-cells has until now hindered an objective evaluation of changes caused by age or experimental stimuli. To overcome this, a rigorous methodology has been designed to detect variations in shape, size, and number of C-cells throughout development. Using this methodology, we have demonstrated that C-cells do not significantly alter their shape with age. However, their volume increases gradually from 472 m³ in newborn rats to 1653 m³ in 120-day-old animals. Over the same time period, the mean number of C-cells within the thyroid gland increased 9-fold (from 1.6x10⁴ to 1.5x10⁵), and the number of C-cells per unit area decreased (from 6.15x10⁴/mm³ to 2.6x10⁴/mm³). We conclude that there are marked variations in size, total number, and number of C-cells per unit area in the rat thyroid gland after birth.     

Effects of prolactin on the morphology and function of rat Leydig cells: short-term versus long-term administration

Summary The bolus administration of prolactin (PRL) to adult rats did not cause any apparent change in the basal and luteinizing hormone (LH)-stimulated blood levels of testosterone (as estimated by radioimmune assay). Prolonged PRL infusion did not affect either basal testosterone plasma concentration or the morphology of Leydig cells (as evaluated by electron microscopy and stereology). Conversely, prolonged PRL treatment notably increased the gonadotrophic effects of chronic LH administration; this mainly consisted of a rise in the blood concentration of testosterone and a conspicuous hypertrophy of Leydig cells. The LH-induced increase in the volume of Leydig cells was the result of an increase in the volumes of all the organelles involved in steroid synthesis (i.e., smooth endoplasmic reticulum, peroxisomes and mitochondria). However, the trophic effects of PRL infusion exclusively concerned smooth endoplasmic reticulum and peroxisomes. In the light of these findings, the hypothesis is advanced that the mechanism underlying the gonadotrophic action of PRL involves an enhancement of the endogenous cholesterol synthesis, which could provide an abundance of precursors for testosterone synthesis, the post-cholesterol steps of which, in turn, would be exclusively controlled by LH.     

Changes in galanin immunoreactivity in rat micturition reflex pathways after cyclophosphamide-induced cystitis

Alterations in the expression of the neuropeptide, galanin, were examined in micturition reflex pathways of rat after cyclophosphamide (CYP)-induced cystitis of variable duration: acute (4 h), intermediate (48 h), or chronic (10 days). In control animals, galanin expression was present in specific regions of the gray matter in the rostral lumbar and caudal lumbosacral spinal cord, including: (1) the dorsal commissure (DCM); (2) superficial dorsal horn; (3) the regions of the intermediolateral cell column (L1–L2) and the sacral parasympathetic nucleus (SPN, L6–S1); and (4) the lateral collateral pathway (LCP) in lumbosacral spinal segments. Densitometry analysis demonstrated significant decreases (P≤0.01) in galanin immunoreactivity (IR) in these regions of the L1–S1 spinal cord after acute or intermediate CYP-induced cystitis. In contrast, increases (P≤0.01) in galanin–IR were observed in the DCM, SPN, or LCP regions in the L6–S1 spinal segments in rats with chronic cystitis. No changes in the number of galanin–immunoreactive cells were observed in the L1–S1 dorsal root ganglia (DRG) after CYP-induced cystitis of any duration. A small percentage of bladder afferent cells (Fast-blue-labeled) in the DRG expressed galanin–IR in control rats; this was not altered with cystitis. Galanin–IR was observed encircling DRG cells after chronic cystitis. These changes may contribute to urinary bladder dysfunction, altered sensation, and referred somatic hyperalgesia after cystitis.This work was supported in part through NIH grants DK051369, DK060481, DK065989, and NS040796.     

Quantification by real-time PCR of developmental and adult myosin mRNA in rat muscles

A real-time RT-PCR assay using newly designed primers was developed to analyze developmental and adult MHC mRNA expression both in skeletal muscles and single fibers. Only 4 ng of total RNA was necessary for the analysis of the relative mRNA expression of MHC genes. Different validation steps were realized concerning both specificity and sensitivity of each primer set, and linearity and efficiency of each real-time PCR amplification. Then, quantification of MHC mRNA in neonatal and adult muscles as well as in single fibers was done by the ΔC_T method, with CycA gene as the reference gene. Due to a higher sensitivity than that of a competitive PCR method, we demonstrated that this assay is suitable to study very low level of MHC mRNA expression as developmental MHC in adult muscle and to quantify mRNA from very small samples.     

Junctions between pericytes and the endothelium in rat myocardial capillaries: A morphometric and immunogold study

Summary Pericytes are cells of mesodermal origin which are closely associated with the microvasculature. Despite numerous studies little is known about their function. We have studied the relationship between pericytes and the endothelium in rat myocardial capillaries employing ultrastructural and immunogold techniques. 14% of the subendothelial cell membrane is covered by comparatively small pericytic cell processes. About half of these processes are completely embedded in baseement membrane material, whereas the remaining half forms closer contacts with the endothelium. These contacts are devoid of anti-laminin immunogold label, a marker for basement membranes. A small fraction of these contacts has been identified as tight junctions resembling those seen between endothelial cells in capillaries of the same tissue. The remaining majority of junctions reveals a cleft of approximately 18 nm between the apposed membranes in which a succession of cleft-spanning structures can often bedetected. It was also found that pericytic processes are preferentially located close to interendothelial junctions. We suggest that the high frequency of intimate junctions between pericytes and the endothelium and the preferential localisation near paracellular clefts may have functional significance.     

Expression of nestin,desmin and vimentin in intact and regenerating muscle spindles of rat hind limb skeletal muscles

We describe the expression and distribution patterns of nestin, desmin and vimentin in intact and regenerating muscle spindles of the rat hind limb skeletal muscles. Regeneration was induced by intramuscular isotransplantation of extensor digitorum longus (EDL) or soleus muscles from 15-day-old rats into the EDL muscle of adult female inbred Lewis rats. The host muscles with grafts were excised after 7-, 16-, 21- and 29-day survival and immunohistochemically stained. Nestin expression in intact spindles in host muscles was restricted to Schwann cells of sensory and motor nerves. In transplanted muscles, however, nestin expression was also found in regenerating “spindle fibers”, 7 and 16 days after grafting. From the 21st day onwards, the regenerated spindle fibers were devoid of nestin immunoreactivity. Desmin was detected in spindle fibers at all developmental stages in regenerating as well as in intact spindles. Vimentin was expressed in cells of the outer and inner capsules of all muscle spindles and in newly formed myoblasts and myotubes of regenerating spindles 7 days after grafting. Our results show that the expression pattern of these intermediate filaments in regenerating spindle fibers corresponds to that found in regenerating extrafusal fibers, which supports our earlier suggestion that they resemble small-diameter extrafusal fibers.     

Distribution of neurons in the major pelvic ganglion of the rat which supply the bladder,colon or penis

Summary In male rats a large number of the postganglionic neurons which innervate the pelvic organs are located in the major pelvic ganglion. In the present study we have identified the location within this ganglion of neurons which project to either of three pelvic organs, the penis, colon or urinary bladder. Two fluorescent retrogradely-transported dyes, Fast Blue and Fluoro-Gold, were used. For most animals one dye was injected into the cavernous space of the penis, the wall of the distal colon or the wall of the urinary bladder. In a small number of animals two organs were injected, each with a different dye. One to six weeks after injection the major pelvic ganglia were fixed in buffered formaldehyde. The distribution of fluorescent dye-labelled cells was observed in whole mounts of complete ganglia and, in most cases, also in small accessory ganglia located between the ureter and the prostate. The studies showed a unique pattern of distribution for each organ-specific group of neurons. Most of the colon neurons are located in the major pelvic ganglion near the entrance of the pelvic nerve, whereas almost all of the penis neurons are near or within the penile nerve. Bladder neurons are relatively evenly distributed throughout the ganglion. These results demonstrate a distinct topographical organization of organ-specific neurons of the major pelvic ganglion of the male rat, a phenomenon which has also been observed in other peripheral ganglia.     

The distribution and origin of nerve fibers immunoreactive for substance P and neurokinin A in the anterior buccal gland of the rat

The distribution and origin of substance P (SP) and neurokinin A (NKA) were studied in rat in the anterior buccal glands, which are minor mucous salivary glands. Indirect immunofluorescence staining showed moderate SP and NKA innervation of salivary acini and interlobular ducts, whereas blood vessels were more sparsely innervated, and there were few nerve fibers in the stroma and around the intralobular ducts. About 10%–20% of the trigeminal ganglion cells showed equally strong immunoreactivity to both SP and NKA. Unilateral denervation of the branches of the trigeminal nerve caused complete disappearance of the stromal fibers and greatly reduced the number of all other SP-immunoreactive and NKA-immunoreactive nerve fibers. In the superior cervical ganglia, SP and NKA immunoreactivity was restricted to small intensely fluorescent cells; SP and NKA immunoreactivity was absent from principal ganglionic cells, and thus sympathectomy had no any effect on the number or distribution of fibers immunoreactive for SP and NKA in the anterior buccal glands. The fibers remaining after sensory denervation could have been of parasympathetic origin, indicating a dual origin of nerves immunoreactive for SP and NKA in these glands. The present data demonstrate that the major part of the glandular SP and NKA innervation in the minor salivary glands derives from the trigeminal ganglia. The distribution of the peripheral nerve fibers indicates that they may play a role in the delivery of potent neuropeptides involved in the vascular, secretory, and motor (myoepithelial cells) functions of salivary glands.     

The cytoarchitecture of the medial layer in rat thoracic aorta: a scanning electron-microscopic study

Summary The cytoarchitecture of the medial layer of rat thoracic aorta was examined by scanning electron microscopy after removal of the connective tissue. The outermost lamella showed a lattice-like structure of muscle bundles of closely apposed smooth muscle cells (SMCs), whereas the inner lamellae consisted of more-or-less continuous muscle sheets of vaguely defined subgroups of parallel SMCs. Longitudinal rows of ridges ran along the adventitial surface of these muscle bundles and sheets. The SMCs of the outermost lamella, were 5.1 m wide, and varied in shape, whereas those of the inner lamellae, were 52.7 m long, 2.6 m wide and 4.1 m thick, and were elongated, spindle-shaped cells with serrated outlines. These latter SMCs extended obliquely, and partially overlapped each other. The surface of the SMCs in the outermost lamella exhibited a rugged texture, with nodular protrusions and oblique and longitudinal laminar folds, while the inner lamellar cells showed longitudinal laminar folds and finger-like processes on both sides of the ridges, pointing in opposite directions to the ridges. The angle of deviation from the transverse axis of the vessel, of the muscle bundles and subgroups in the outermost lamella, was 33.6°, in the second and third lamellae, 22.5°, and in the innermost lamellae, 12.8°. The mean angle of the muscle bundle and subgroup arrangement, with respect to the long axis of the vessel, however, was basically 90° in all lamellae.     

Ultrastructural immunolocalization of IGF-1 and insulin receptors in rat pituitary culture: evidence of a functional interaction between gonadotroph and lactotroph cells

We have investigated the expression of receptors for insulin and insulin-like growth factor 1 (IGF-1) in rat pituitary cells in vitro and examined the morphological and proliferative changes induced in adenohypophyseal cells by insulin and IGF-1. The proliferation of lactotrophs was determined by double-immunostaining for bromodeoxyuridine and prolactin. Incubation with insulin (10, 100 or 1000 ng/ml) or IGF-1 (5, 30 or 100 ng/ml) for 48 or 72 h significantly increased the number of lactotrophs undergoing mitosis. Co-incubation of insulin or IGF-1 with genistein (25 μM), an inhibitor of the tyrosine kinase receptor, reduced the proliferation of lactotrophs elicited by the hormone and the growth factor. The receptors for insulin and IGF-1 were localized in intact pituitary cells by ultrastructural immunocytochemistry with the colloidal gold-protein A technique. Gonadotrophs expressed both receptors, specific labelling being restricted to this cell type. Electron-microscopical observations of pituitary cell cultures incubated with insulin or IGF-1 revealed gonadotroph cells exhibiting the fine-structural features of enhanced protein synthetic activity. These findings suggest that both insulin and IGF-1 are able to induce the proliferation of lactotrophs through an indirect mechanism mediated by a factor synthesized by gonadotroph cells, in addition to stimulating the biosynthetic activity of the gonadotroph in a direct manner.This work was supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) and the Secretaría de Ciencia y Tecnología de la Universidad Nacional de Córdoba (SECyT).     

Immunocytochemical localization of aromatase-containing neurons in the rat brain during pre- and postnatal development

the present immunohistochemical study demonstrates the ontogenetic appearance of aromatase-immunoreactive neurons in several discrete regions of the hypothalamus and limbic system in the rat brain, using a purified antibody against human placental aromatase cytochrome P450. Immunoreactive cells were first detected in the preoptic area on the 13th day of embryonic life (E 13), and additionally in the bed nucleus of the stria terminalis on E 15. Labeled cells were also found in the medial amygdaloid nucleus and the ventromedial nucleus on E 16, and some were detected in the arcuate nucleus on E 19. As gestation progressed, the number and the immunoreactivity of these cells gradually increased and peaked within definite periods of perinatal life and there-after declined or disappeared. The immunoreactive cells were also found in the central amygdaloid nucleus and the lateral septal nucleus, and in the ventral pallidum, after the 14th day of postnatal life (P 14) and 30th day (P 30), respectively. The distribution of aromatase-immunoreactive neurons was similar between the sexes, while the immunoreactivity was higher in males than in females after late gestational days. No immunoreaction was detectable in other regions of the telencephalon or midbrain at any time periods studied. The aromatase-immunoreactive neurons in the specific regions may be involved in the sexual differentiation of the brain.     

Doxazosin reduces cell proliferation and increases collagen fibers in rat prostatic lobes

We investigated the effects of doxazosin (Dox), an alpha-adrenoceptor antagonist used clinically for the treatment of benign prostatic hyperplasia (BPH), on the rat prostatic complex by assessing structural parameters, collagen fiber content, cell proliferation, and apoptosis. Adult Wistar rats were treated with Dox (25 mg/kg per day), and the ventral (VP), dorsolateral, and anterior prostate (AP) regions of the prostate complex were excised at 3, 7, and 30 days after treatment. At 24 h before being killed, the rats were injected once with 5-bromodeoxyuridine (BrdU; thymidine analog) to label mitotically active cells. The prostates were weighed and processed for histochemistry, morphometry-stereology, immunohistochemistry for BrdU, Western blotting for proliferating cell nuclear antigen (PCNA), and the TUNEL reaction for apoptosis. Dox-treated prostate lobes at day 3 presented increased weight, an enlarged ductal lumen, low cubical epithelial cells, reduced epithelial folds, and stretched smooth muscle cells. However, at day 30, the prostates exhibited a weight reduction of ∼20% and an increased area of collagen and reticular fibers in the stromal space. Dox also reduced epithelial cell proliferation and increased apoptosis in the three prostatic lobes. Western blotting for PCNA confirmed the reduction of cell proliferation by Dox, with the AP and VP being more affected than the dorsal prostate. Thus, Dox treatment alters epithelial cell behavior and prostatic tissue mechanical demand, inducing tissue remodeling in which collagen fibers assume a major role. This work was funded by FAPESP (Sao Paulo State Research Foundation; grants 04/13261-8, to S.L.F.), by FUNDUNESP (Foundation for Development of Sao Paulo State University), and by CNPq (Brazilian National Research and Development Council; fellowship to L.A.J. Jr). This paper represents part of the PhD thesis presented by L.A.J. Jr to the State University of Campinas - UNICAMP, Brazil.     

Effects of denervation on the composition and synthesis of sarcolemmal proteins

In vitro synthesis of proteins and changes in polypeptide composition of sarcolemma were studied in innervated and denervated extensor digitorum longus muscle of the rat. A technique of evacuating myoplasm from muscle slices was used as a preliminary step in the preparation of three membrane fractions, M, H and S, containing sarcolemma. On the basis of findings from the previous study and the present investigation, it was concluded that the M fraction was most enriched with extrajunctional sarcolemma.In vitro incorporation of [³H]leucine into membrane proteins of the M fraction showed an apparent linear increase in the rate of protein synthesis from 1–10 days after denervation. The relative increase at 10 days was 137% greater than that of innervated controls. Fractions H and S showed a smaller relative increase.Polypeptide composition of M, H and S fractions based on SDS gel electrophoresis of innervated and denervated muscle, showed qualitative and quantitative changes. The most striking difference was a nominal 29 000 component in M that constituted a disproportionately large peak. Following 10 days of denervation the M fraction underwent significant compositional changes in its electrophoretic profile, the most dramatic of which was a large reduction in the proportion of the 29 000 component. The denervation-induced compositional change is discussed in light of known alterations in the chloride conductance of the muscle plasmalemma.