Consequences of ankle joint immobilisation: insights from a morphometric analysis about fibre typification, intramuscular connective tissue, and muscle spindle in rats.

Orthosis immobilisations are routinely used in orthopaedic procedures. This intervention is applicable in bone fractures, ligament injuries, and tendonitis, among other disorders of the musculoskeletal system. We aimed to evaluate the effects of ankle joint functional immobilisation on muscle fibre morphology, connective tissue, muscle spindle and fibre typification triggered by a novel metallic orthosis. We developed a rodent-proof experimental orthosis able to hold the tibiotalar joint in a functional position for short and long terms. The tibialis anterior muscles of free and immobilised legs were collected and stained by histology and histochemistry techniques to investigate general muscle morphology, connective tissue and muscle fibre typification. Morphometric analysis of muscle cross-section area, fibre type cross-section area, fibre type density, percentage of intramuscular connective tissue, and thickness of the muscle spindle capsule were obtained to gain insights into the experimental protocol. We found that short- and long-term immobilisation decreased the cross-section area of the muscles and induced centralisation of myonuclei. The connective tissue of immobilised muscle increased after 2 and 4 weeks mainly by deposition of type III and type I collagen fibres in the perimysium and endomysium, respectively, in addition to muscle spindle capsule thickening. Type IIB muscle fibre was severely affected in our study; the profile assumed odd shapes, and our data suggest interconversion of these fibre types within long-term immobilisation. In conclusion, our protocol has produced structural and histochemical changes in muscle biology. This method might be applied to various rodent models that enable genetic manipulation for the investigation of muscle degeneration/regeneration processes.

Expression of degenerative markers in intervertebral discs of young and elderly asymptomatic individuals.

Intervertebral disc (IVD) degeneration is a remodeling process mediated by several growth factors and cytokines. This process has been extensively studied in vitro and with pathologic specimens obtained during surgery for scoliosis or back pain. However, the occurrence and temporal evolution of these molecules during normal aging, particularly in the cervical segment, is not known. Our objective was to study and compare the presence of putative mediators in the IVD of young (<35 years, G1) and elderly (>65 years, G2) presumably asymptomatic individuals. Thirty C4-5 and C5-6 discs and thirty L4-5 and L5-S1 discs per group were collected during the autopsy of individuals whose family members denied a history of neck or back pain. Discs were divided into anterior, central (lumbar only) and posterior sectors for analysis. Immunohistochemistry for TNF-α, IL-1ß, VEGF, NGF-ß, BDNF, TIMP-1, MMP-1, -2 and -3 was performed and reactivity compared between groups and sectors. All of these molecules were detected in every disc sector of both G1 and G2. Most statistical comparisons (25/45, 55.6%) revealed an increase in mediator expression in G2 in relation to G1. Regional differences in the expression of remodeling enzymes were rare; NGF-ß and BDNF had slightly higher expression in the cervical segment of elderly individuals. A senescent profile with elevated VEGF, MMP-2 and MMP-3 was observed across most G2 disc regions and were generally elevated from G1. In conclusion, the mere presence of any of the studied molecules inside the IVD cannot be considered pathologic. Expression of remodeling enzymes and inflammatory mediators is relatively similar across different vertebral segments and disc regions leading to a common degenerated pattern, while neurotrophins have slightly higher expression in cervical discs. These findings support the concept that disc remodeling in different segments follows a similar pathway that can be potentially mediated to avoid structural failure.

Normal aging in human lumbar discs: An ultrastructural comparison.

The normal aging of the extracellular matrix and collagen content of the human lumbar intervertebral disc (IVD) remains relatively unknown despite vast amounts of basic science research, partly because of the use of inadequate surrogates for a truly normal, human IVD. Our objective in this study was to describe and compare the morphology and ultrastructure of lumbar IVDs in 2 groups of young (G1-<35 years) and elderly (G2->65 years). Thirty L4-5 and L5-S1 discs per group were obtained during autopsies of presumably-asymptomatic individuals and analyzed with magnetic resonance imaging (MRI), a morphological grading scale, light microscopy, scanning electron microscopy (SEM) and immunohistochemistry (IHC) for collagen types I, II, III, IV, V, VI, IX and X. As expected, a mild to moderate degree of degeneration was present in G1 discs and significantly more advanced in G2. The extracellular matrix of G2 discs was significantly more compact with an increase of cartilaginous features such as large chondrocyte clusters. Elastic fibers were abundant in G1 specimens and their presence correlated more with age than with degeneration grade, being very rare in G2. SEM demonstrated persistence of basic structural characteristics such as denser lamellae with Sharpey-type insertions into the endplates despite advanced age or degeneration grades. Immunohistochemistry revealed type II collagen to be the most abundant type followed by collagen IV. All collagen types were detected in every disc sector except for type X collagen. Statistical analysis demonstrated a general decrease in collagen expression from G1 to G2 with an annular- and another nuclear-specific pattern. These results suggest modifications of IVD morphology do not differ between the anterior or posterior annulus but are more advanced or happen earlier in the posterior areas of the disc. This study finally describes the process of extracellular matrix modification during disc degeneration in an unselected, general population and demonstrates it is similar to the same process in the cervical spine as published previously.

Morphological effects of autoclaved diet on the myenteric neurons of rats.

AIM: To evaluate the effect of autoclaved diet on the jejunum neurons of the myenteric plexus of rats during their growth. METHODS: The experimental groups were made up of rats going through weaning whose mothers received either an autoclaved or a non-autoclaved diet during gestation and lactation, and rats that were fed the same diet as their mothers during the post-weaning period. In order to measure the neurons' body profile and to quantify the number of neurons per area, preparations were stained by the nicotinamide adenine dinucleotide-diaphorase method. RESULTS: No significant changes were observed in rats' body weight or in the number of neurons regardless of the diet used (P > 0.05). There was a decrease in the jejunum-ileum length in rats treated with an autoclaved diet (P < 0.05). An increase in the neuronal cross-sectional area was seen in rats that had received the autoclaved diet, an effect that was significant for animals undergoing weaning. In addition, all observed factors showed significant differences when related to the age of the animals. CONCLUSION: The autoclaved diet did not alter the quantity of neurons, but increased their cell body area, suggesting changes similar to those observed in protein deficiency.

Estimating the length density of convoluted tubular systems - II: comparative analysis using five different methods.

An artificial convoluted tubular system with precise predefined parameters was created. It was stereologically analyzed in order to preview the potential probability to introduce errors when applied to biological systems like testicular tubes or glomerule like structures. The length of the convoluted tubules more frequently analyzed was estimated by five different methods. The analytical methods were based on both the number of tubule transections, which is related to the transection area and/or organ volume, and the axis length of an ideal cylinder. Tubular systems were analyzed with or without consideration of the transection shape. When shape was considered, two methods were compared: one evaluating the major and minor axes from elliptical profiles, and the other the crosses between parallel lines of a test system superimposed on circular, elliptical, or more complex profiles. Comparison of the five methods revealed different estimations of the length in relation to the pre-determined model, which varied from an 11.8% overestimation to a 39% underestimation. The fractionator method was proposed as alternative to diminish the work overload when counting intersections between lines of test systems and transection profiles. The results with the fractionator are very promising concerning the application of the method in laboratories of pathology.

Effects of perinatal protein deprivation and recovery on esophageal myenteric plexus.

AIM: To evaluate effects of pre- and postnatal protein deprivation and postnatal recovery on the myenteric plexus of the rat esophagus. METHODS: Three groups of young Wistar rats (aged 42 d) were studied: normal-fed (N42), protein-deprived (D42), and protein-recovered (R42). The myenteric neurons of their esophagi were evaluated by histochemical reactions for nicotinamide adenine dinucleotide (NADH), nitrergic neurons (NADPH)-diaphorase and acetylcholinesterase (AChE), immunohistochemical reaction for vasoactive intestinal polypeptide (VIP), and ultrastructural analysis by transmission electron microscopy. RESULTS: The cytoplasms of large and medium neurons from the N42 and R42 groups were intensely reactive for NADH. Only a few large neurons from the D42 group exhibited this aspect. NADPH detected in the D42 group exhibited low reactivity. The AChE reactivity was diffuse in neurons from the D42 and R42 groups. The density of large and small varicosities detected by immunohistochemical staining of VIP was low in ganglia from the D42 group. In many neurons from the D42 group, the double membrane of the nuclear envelope and the perinuclear cisterna were not detectable. NADH and NADPH histochemistry revealed no group differences in the profile of nerve cell perikarya (ranging from 200 to 400 microm(2)). CONCLUSION: Protein deprivation causes a delay in neuronal maturation but postnatal recovery can almost completely restore the normal morphology of myenteric neurons.

Mastication effort study using photorefractive holographic interferometry technique.

The purpose of this work was the force-displacement response analysis of the masticatory process in a dried human skull by Double-Exposure Photorefractive Holographic Interferometry Technique (2E-PRHI). The load concentration and dissipation of the forces from dried human skull were analysed at applied loading stands as a Simulation of Isolated Contraction (SIC) of some mastication muscles. The 2EHI and Fringe Analysis Method were used to obtain the quantitative results of this force-displacement response. These results document quantitatively the real biomechanical response from dried human skull under applied loading and it can be used for complementary study by finite element model and others analysis type.

Analysis of the brainstem internal morphology of the capuchin monkey (Cebus apella) / Análise da morfologia interna do tronco encefálico de macaco-prego (Cebus apella)

The gross anatomy and the internal morphology of the brainstem of the monkey Cebus apella were described based on the observations made on adult brainstens. Six adult Cebus apella monkeys were used for anatomical observations and for the study of the internal morphology they were cut into serial sections at six different levels (pyramidal decussation; superior portion of the olive; inferior portion of the olive; pons; superior colliculus and inferior colliculus) and properly stained for studies of the internal structure. Our results show that the Cebus apella brainstem is characterized by the presence of a bulbpontine sulcus; an enlarged brainstem, particularly of the medulla oblongata, with a consequent laterization of the trapezoid body, the presence of inferior olivary complex, the apparent origin of cranial nerves distant from the pyramids and of its decussation; presence of the antero-lateral sulcus separating the pyramids of the olives; expressive development of the trapezoid body, of the pons, nuclei of the pons and of the cerebral peduncle. These results are consistent with previous descriptions for other primates and suggest that the organization and development of the Brainstem structures share similarities with superior primates including humans which reflect the motor abilities of the studied species.

A anatomia e a morfologia interna do tronco encefálico (TE) do macaco-prego (Cebus apella) foram descritas com base nas observações feitas em troncos encefálicos de macacos adultos. Seis macacos Cebus apella adultos foram utilizados para as observações anatômicas. Para os estudos da morfologia interna os TE foram cortados seriadamente em seis níveis diferentes: decussação piramidal, porção superior da oliva, porção inferior da oliva, ponte, colículo superior e colículo inferior. Após esse procedimento eles foram corados com a técnica de Mulligan modificada por nós. Nossos resultados mostram que o TE de Cebus apella é caracterizado pela presença do sulco bulbo-pontino, aumento do tamanho do TE, especialmente da medula oblonga, com consequente lateralização do corpo trapezoide, presença do sulco ântero-lateral separando as pirâmides das olivas; desenvolvimento expressivo do corpo trapezoide, da ponte, dos núcleos da ponte e do pedúnculo cerebral. Estes resultados são consistentes com descrições prévias para outros primatas na literatura e sugerem que a organização e desenvolvimento do TE exibe semelhanças com primatas superiores, incluindo o homem, o que reflete as habilidades motoras da espécie estudada.

Morphological and quantitative study of ganglionated plexus of Calomys callosus trachea.

Calomys callosus is a wild, native forest rodent found in South America. In Brazil, this species has been reported to harbour the parasitic protozoan Trypanosoma cruzi. The ganglionated plexus of this species was studied using whole-mount preparations of trachea that were stained using histological and histochemical methods. The histological methods were used to determine the position of the ganglia with respect to the trachea muscle and to determine the presence of elastic and collagen fibers. The histochemical method of NADH-diaphorase was used for morphometric evaluations of the plexus. The tracheal plexus lies exclusively over the muscular part of the organ, dorsal to the muscle itself. It varies in pattern and extent between animals. The average number of neurons was 279 and the cellular profile area ranged from 38.37 microm2 to 805.89 microm2. Acetylcholinesterase (AChE) histochemistry verified that both ganglia and single neurons lie along nerve trunks and are reciprocally interconnected with the plexus. Intensely AChE-reactive neurons were found to be intermingled with poorly reactive ones. Two longitudinal AChE-positive nerve trunks were also observed and there was a diverse number of ganglia along the intricate network of nerves interconnecting the trunks. A ganglion capsule of collagen and elastic fibers surrounding the neurons was observed. Under polarized light, the capsule appeared to be formed by Type I collagen fibers.

Effect of oleic and linoleic acids on the inflammatory phase of wound healing in rats.

Inflammation is a crucial step for the wound healing process. The effect of linoleic and oleic acids on the inflammatory response of the skin during the healing process and on the release of pro-inflammatory cytokines by rat neutrophils in vitro was investigated. A wound in the dorsal surface of adult rats was performed and fatty acids were then topically administered. Both oleic and linoleic acids increased the wound healing tissue mass. The total protein and DNA contents of the wounds were increased by the treatment with linoleic acid. The treatments with oleic and linoleic acids did not affect vascular permeability. However, the number of neutrophils in the wounded area and air pouches was increased and the thickness of the necrotic cell layer edge around the wound was decreased. A dose-dependent increase in vascular endothelial growth factor-alpha (VEGF-alpha) and interleukin-1beta (IL-1beta) by neutrophils incubated in the presence of oleic and linoleic acid was observed. Oleic acid was able to stimulate also the production of cytokine-induced neutrophil chemoattractant in inflammation 2 alpha/beta (CINC-2alpha/beta). This pro-inflammatory effect of oleic and linoleic acids may speed up the wound healing process.

A simple method for estimating the length density of convoluted tubular systems.

We present a new method for estimating the length density (Lv) of convoluted tubular structures exhibiting an isotropic distribution. Although the traditional equation Lv=2Q/A is used, the parameter Q is obtained by considering the collective perimeters of tubular sections. This measurement is converted to a standard model of the structure, assuming that all cross-sections are approximately circular and have an average perimeter similar to that of actual circular cross-sections observed in the same material. The accuracy of this method was tested in eight experiments using hollow macaroni bent into helical shapes. After measuring the length of the macaroni segments, they were boiled and randomly packed into cylindrical volumes along with an aqueous suspension of gelatin and India ink. The solidified blocks were cut into slices 1.0 cm thick and 33.2 cm2 in area (A). The total perimeter of the macaroni cross-sections so revealed was stereologically estimated using a test system of straight parallel lines. Given Lv and the reference volume, the total length of macaroni in each section could be estimated. Additional corrections were made for the changes induced by boiling, and the off-axis position of the thread used to measure length. No statistical difference was observed between the corrected estimated values and the actual lengths. This technique is useful for estimating the length of capillaries, renal tubules, and seminiferous tubules.

Phase-shifting real-time holographic interferometry applied to load transmission evaluation in dried human skull.

Phase-shifting real-time holography with photorefractive Bi(12)SiO(20) crystal as holographic recording medium applied to load transmission evaluation and tension dissipation on a dried human skull under loading is presented. The applied loading stands as a simulation of isolated contraction (SIC) of some masticatories muscles. The four-frames phase-shifting technique and the unwrapping branch-cut technique were used to obtain the phase map. The quantitative results show the feasibility of the employed system in the study of microdisplacements in the skull structure provided by SIC.

Effects of combined pre- and post-natal protein deprivation on the myenteric plexus of the esophagus of weanling rats: a histochemical, quantitative and ultrastructural study.

AIM: To evaluate the effects of protein deprivation on the myenteric plexus of the esophagus of weanling rats. METHODS: Pregnant female Wistar rats were divided into 2 groups: nourished (N), receiving normal diet, and undernourished (D), receiving a protein-deprived diet, which continued after birth. At twenty-one days of age, 13 esophagi from each group were submitted to light microscopy and morphometrical analysis employing the NADH diaphorase, NADPH diaphorase and acetylcholinesterase techniques. Three other esophagi from each group were evaluated with transmission electron microscopy (TEM). RESULTS: In both the NADH- and the NADPH-reactive mounts, the neurons of the N mounts were more intensely stained, while in the D esophagi only the larger neurons were reactive. Many myenteric neurons of N were intensely reactive for AChE activity but only a few neurons of D exhibited these aspects. Ultrastructural analysis revealed that the granular reticulum of N showed large numbers of ribosomes aligned on the outer surface of its regularly arranged membrane while the ribosomes of D were disposed in clusters. The chromatin was more homogeneously scattered inside the neuron nucleus of N as well as the granular component of the nucleolus was evidently more developed in this group. Statistically significant differences between N and D groups were detected in the total estimated number of neurons stained by the NADPH technique. CONCLUSION: The morphological and quantitative data shows that feeding with protein-deprived diet in 21-d old rats induces a delay in the development of the myenteric neurons of the esophagus.

Basilar expansion of the human sphenoidal sinus: an integrated anatomical and computerized tomography study.

Basilar expansion of the sphenoidal sinus (BESS) was studied in order to demonstrate its critical relevance in endoscopic or microscopic endonasal surgical interventions, including access to the sphenoidal sinus itself or in transsphenoidal pituitary approaches. Direct evaluation of anatomical specimens (25 dry skulls and 25 formalin-fixed hemi heads) and the use of computerized tomography (CT) (50 dry skulls and 750 patients) showed a high BESS frequency (69%). The authors considered BESS to be critical when the posterior wall of the clivus was 2-mm thick and found a high incidence of this important anatomical variation (44%). This study also evaluated the relationship between the sinonasal septa, the clivus, and the internal carotid arteries, and a considerable regularity in the location of these structures was seen. The septa were anatomically related to the internal carotid arteries in 55% and to the clivus in 33% of the cases. In conclusion, the high frequency of critical BESS here described is relevant to endoscopic or microscopic endonasal surgical interventions, including access to the sphenoidal sinus itself or in transsphenoidal pituitary approaches.

Ultrastructural study of the filum terminale and its elastic fibers.

OBJECTIVE: The filum terminale (FT) is a fibrovascular band involved in the pathophysiology of tethered cord syndrome (TCS). Its morphological and ultrastructural properties remain largely unknown even though they are thought to play a role in the generation of TCS in adult patients with normal level conus medullaris. MATERIALS AND METHODS: Twenty fresh adult human cadavers had their fila measured and removed. Transversal and longitudinal sections of the proximal, middle, and distal thirds of FT were submitted to light microscopy analysis with four different techniques. Five fila were selected for longitudinal and transversal scanning electron microscopy analysis. RESULTS: The bulk of the FT is composed of 5- to 20-microm thick longitudinal bundles of Type 1 collagen separated by 3- to 10-microm intervals, although capillaries and other elements may be present. A delicate (0.05-1.5 microm) meshwork of predominantly Type 3 collagen transversal fibers connects these bundles. Abundant longitudinally oriented elastic and elaunin fibers are found inside collagen bundles. A complex tridimensional structure is evidenced on electron microscopy. CONCLUSION: The longitudinal arrangement of collagen bundles and the impressive amount of elastic and elaunin fibers should elicit considerable elastic properties to the FT. An altered elasticity mechanism has been proposed for TCS; further studies are needed with TCS patients to define whether the collagen structure, Type 1/Type 3 proportion, or elastic fiber content are altered, which could lead to new histopathological definitions of TCS, helping neurosurgeons in the difficult management of TCS patients with normal level conus medullaris.

The innervation of the thymus gland in the rat: a HRP study

The marker horseradish peroxidase (HRP) was used to retrogradely label the cell bodies of origin of the nerve fibers responsible for thymic innervation. The thymus was exposed in albino rats under general anesthesia and a conjugate of HRP-WGA was injected bilaterally into the gland parenchyma. After a survival time of 3 days, the animals were anesthetized and perfused transcardially. Their brainstems were removed and serially sectioned and together with the whole sympathetic chains were processed for HRP demonstration. Labeled cell bodies were identified in the brainstem at the level of the retrofacial and ambiguous nuclei bilaterally. In the sympathetic chain, labeled neurons were present from the first cervical to the ninth thoracic ganglion, although they were detected in higher numbers in the superior cervical and stelate ganglia.

A quantitative study of the myenteric plexus of the distal end of the human esophagus

The density of ganlia and nerve cells in the myenteric plexus of the last distal (12 cem. of the human esophagus was determined in six esophagi from autops y material. The density of ganglia and nerve cells in cresyl violet stained stretch preparations of twelve esophageal segments, each 1 cm. in length, were compared. The highest values were recorded in segments 4,5 and 6 cm. above the cardiac incisura. The lowest densities of ganglia and nerve cells were found in segments 1,2 and 3 cm. above the cardiac incisura. The segments contained 659-3316 perikarya/cm2; ganglia contained 3-310 cells