Morphology and morphometry of the ulnar nerve in the forelimb of pigs.

The knowledge of the morphology and morphometry of peripheral nerves is essential for developing neural interfaces and understanding nerve regeneration in basic and applied research. Currently, the most adopted animal model is the rat, even though recent studies have suggested that the neuroanatomy of large animal models is more comparable to humans. The present knowledge of the morphological structure of large animal models is limited; therefore, the present study aims to describe the morphological characteristics of the Ulnar Nerve (UN) in pigs. UN cross-sections were taken from seven Danish landrace pigs at three distinct locations: distal UN, proximal UN and at the dorsal cutaneous branch of the UN (DCBUN). The nerve diameter, fascicle diameter and number, number of fibres and fibre size were quantified. The UN diameter was larger in the proximal section compared to the distal segment and the DCBUN. The proximal branch also had a more significant number of fascicles (median: 15) than the distal (median: 10) and the DCBUN (median: 11) segments. Additionally, the mean fascicle diameter was smaller at the DCBUN (mean: 165 µm) than at the distal (mean: 197 µm) and proximal (mean: 199 µm) segments of the UN. Detailed knowledge of the microscopical structure of the UN in pigs is critical for further studies investigating neural interface designs and computational models of the peripheral nervous system.

Effects of Low-Level Laser Therapy and Purified Natural Latex (Hevea brasiliensis) Protein on Injured Sciatic Nerve in Rodents: Morpho-Functional Analysis.

The present study analyzed the effects of low-level laser therapy (LLLT) and the purified natural latex protein (Hevea brasiliensis, F1 protein) on the morpho-function of sciatic nerve crush injuries in rats. One-hundred and eight male Wistar rats were randomly allocated to six groups (n = 18): 1. Control; 2. Exposed (nerve exposed); 3. Injury (injured nerve without treatment); 4. LLLT (injured nerve irradiated with LLLT (15 J/cm2, 780 nm)); 5. F1 (injured nerve treated with F1 protein (0.1%)); and 6. LLLT + F1 (injured nerve treated with LLLT and F1). On the 1st, 7th, 14th, and 56th days after injury, a functional sensory analysis of mechanical allodynia and mechanical hyperalgesia and a motor analysis of grip strength and gait were performed. After 3, 15, and 57 days, the animals were euthanized for morphometric/ultrastructural analyses. The treatments applied revealed improvements in morphometric/ultrastructural parameters compared to the injured group. Sensory analyses suggested that the improvements observed were associated with time progression and not influenced by the treatments. Motor analyses revealed significant improvements in grip strength from the 7th day in the LLLT group and in gait from the 56th day in all treated groups. We concluded that even though the morphological analyses showed improvements with the treatments, they did not influence sensory recovery, and LLLT improved motor recovery.

Histological and Immunohistochemical Analysis of the Effects of Topical Melatonin Treatment Associated with Collagen Sponge and rhBMP-2 Protein on Bone Remodeling.

Extensive bone defect healing is an important health issue not yet completely resolved. Different alternative treatments have been proposed but, in face of a critical bone defect, it is still very difficult to reach a complete regeneration, with the new-formed bone presenting all morphological and physiological characteristics of a normal, preinjury bone. Topical melatonin use has shown as a promising adjuvant for bone regeneration due to its positive effects on bone metabolism. Thus, to search for new, safe, biological techniques that promote bone repair and favor defect healing, we hypothesized that there is a synergistic effect of melatonin treatment associated with rhBMP-2 to guide bone regeneration. This study aimed to investigate bone repair effects of topical melatonin administration in different concentrations (1, 10, and 100 µg), associated or not with rhBMP-2. Surgical-induced bone defect healing was qualitatively evaluated through histopathological analysis by light microscopy. Additionally, quantitative stereology was performed in immunohistochemistry-prepared tissue to identify angiogenic, osteogenic, and osteoclastogenic factors. Quantification data were compared between groups by the ANOVA/Tukey test and differences were considered significant when p < 0.05. Our results showed that the presence of the scaffold in the bone defect hindered the process of bone repair because in the group treated with "blood clot + scaffold" the results of bone formation and immunolabeling were reduced in comparison with all other groups (treated with melatonin alone or in association with rhBMP-2). Statistical analysis revealed a significant difference between the control group (bone defect + blood clot), and groups treated with different concentrations of melatonin in association with rhBMP-2, indicating a positive effect of the association for bone repair. This treatment is promising once it becomes a new safe alternative technique for the clinical treatment of fractures, bone defects, and bone grafts. Our results support the hypothesis of the safe use of the association of melatonin and rhBMP-2 and have established a safe and effective dose for this experimental treatment.

Revisão de anatomia e correlações clínicas do tronco encefálico, parte i: bulbo / Review of brain anatomy and clinical correlations, part i: medulla

O Tronco encefálico (TE) é uma estrutura singular do sistema nervoso central, pois nele passam tratos sensoriais ascendentes da medula espinal, tratos sensoriais da cabeça e do pescoço, os tratos descendentes motores originados no prosencéfalo (divisão mais rostral do encéfalo), e as vias ligadas aos centros de movimento dos olhos. Contém ainda os núcleos dos nervos cranianos e está envolvido na regulação do nível de consciência através de projeções ao prosencéfalo oriundas da formação reticular. Todas essas estruturas coexistem em um espaço muito exíguo, o que faz com que o TE seja um local muito sensível às alterações patológicas, sendo que os pacientes apresentam muitos sinais neurológicos mesmo com lesões muito pequenas nesse local. Compreender a anatomia interna do TE é essencial para o diagnóstico neurológico e a prática da medicina clínica. Outros profissionais da saúde também se beneficiam desse conhecimento para melhor manejo dos seus pacientes neurológicos. Essa revisão apresenta detalhes da anatomia macroscópica e microscópica do bulbo, bem como seus correlatos clínicos frente às lesões mais comuns dessa divisão particular do TE, conhecidas como síndromes bulbares.

The brainstem is a unique structure in the central nervous system, since it gives way to ascending sensory tracts from the spinal cord, sensory tracts from the head and neck, motor descending tracts originating from the forebrain, and the pathways connected to the eye movement centers. It also contains the cranial nerve nuclei and is involved in the regulation of consciousness levels through projections to the forebrain originating in the reticular formation. All these structures coexist in a very small space, which makes the brainstem very sensitive to pathological changes, with patients presenting several neurological symptoms even with very small brainstem lesions. Understanding the internal anatomy of the brainstem is essential for neurological diagnosis and the practice of clinical medicine. Other health professionals also benefit from this knowledge to better manage their neurological patients. This review presents detailed information on the macroscopic and microscopic anatomy of the medulla, as well as its clinical correlates in the face of the most common lesions of this particular division of the brainstem, known as medullary syndromes.

Standardized statement for the ethical use of human cadaveric tissues in anatomy research papers: Recommendations from Anatomical Journal Editors-in-Chief.

Human cadaveric donors are essential for research in the anatomical sciences. However, many research papers in the anatomical sciences often omit a statement regarding the ethical use of the donor cadavers or, as no current standardized versions exist, use language that is extremely varied. To rectify this issue, 22 editors-in-chief of anatomical journals, representing 17 different countries, developed standardized and simplified language that can be used by authors of studies that use human cadaveric tissues. The goal of these editor recommendations is to standardize the writing approach by which the ethical use of cadaveric donors is acknowledged in anatomical studies that use donor human cadavers. Such sections in anatomical papers will help elevate our discipline and promote standardized language use in others non anatomy journals and also other media outlets that use cadaveric tissues.

Morphometric analysis of nerve fibers in neural leprosy.

BACKGROUND: The present study assesses the contributions of axonal degeneration and demyelination in leprosy nerve damage. New clinical strategies can emerge from an in-depth understanding of the pathogenesis of neural leprosy (NL). METHODS: Morphometric analysis of myelinated nerve fibers was performed on 44 nerve biopsy samples collected from leprosy patients. Measures of density, diameter distribution, g-ratios, and the counting of axonal ovoids on the myelinated fibers were taken and compared to those in the control group. RESULTS: The proportion of small myelinated fibers increased in the leprosy group while large fiber frequency decreased. Indicative of axonal atrophy, the g-ratio was lower in the leprosy group. The frequency of axonal ovoids was identical to that found in the non-leprosy neuropathies. CONCLUSIONS: Axonal atrophy, Wallerian degeneration, and demyelination coexist in NL. Axonal degeneration predominates over demyelination in the chronic course of the disease; however, this may change during leprosy reactive episodes. This study regards demyelination and axon degeneration as concurrent mechanisms of damage to nerve fibers in leprosy. It also calls into question the view that demyelination is the primary and predominant mechanism in the complex pathogeny of NL.

Expression of AIF, PARP, and miRNAs MIR-145, MIR-210, and MIR-486 associated with apoptosis in the corpus cavernosum of rats subjected to chronic alcoholism model / Expresión de AIF, PARP y miRNAs MIR-145, MIR-210 y MIR-486 asociados con apoptosis en el cuerpo cavernoso de ratas sometidas a modelo de alcoholismo crónico

SUMMARY: Previous studies from our group described the consequences of using ethanol on penile erection. Nevertheless, the molecular mechanisms surrounding microRNAs, apoptosis process and their relationship with erectile dysfunction associated with alcohol consumption are still poorly understood. The objective of this analysis was to evaluate the mechanism of apoptosis by the expression of AIF and PARP, as well as their regulatory microRNAs: miR-145, miR-210 and miR-486, in the corpus cavernosum of rats submitted to a semivoluntary alcoholism model. For this study 24 Wistar rats were divided into two groups: control (C) and treated with 20 % ethanol (A) for seven weeks. The corpus cavernosum samples were prepared for immunohistochemical analysis of AIF and PARP protein expression, and microRNAs miR-145, miR-210, miR-486 gene expression in cavernous tissue was performed by real time PCR. The immunohistochemical analysis showed little nuclear positive labeling for the protein PARP and AIF in the corpus cavernosum of control and ethanol treated animals. After analysis of miR-145, -210 and -486 microRNA expression in the 12 animals studied, no results were found with significant statistical difference between the control and alcoholized groups. The expression of AIF and PARP and their regulatory microRNAs involved in apoptotic process (miR-145, miR-210 and miR-486) were not altered in the corpus cavernosum of rats submitted to semivoluntary alcoholism.

RESUMEN: Estudios previos de nuestro grupo describieron las consecuencias del uso de etanol en la erección del pene. Sin embargo, los mecanismos moleculares que rodean a los microARN, el proceso de apoptosis y su relación con la disfunción eréctil asociada con el consumo de alcohol aún no se conocen bien. El objetivo de este análisis fue evaluar el mecanismo de apoptosis mediante la expresión de AIF y PARP, así como sus microARN reguladores: miR-145, miR-210 y miR-486, en el cuerpo cavernoso de ratas sometidas a un modelo de alcoholismo semivoluntario. Se dividieron 24 ratas Wistar en dos grupos: control (C) grupo de ratas tratadas con etanol al 20 % (A) durante siete semanas. Las muestras del cuerpo cavernoso se prepararon para el análisis inmunohistoquímico de la expresión de la proteína AIF y PARP, y la expresión del gen microRNAs miR-145, miR-210, miR-486 en tejido cavernoso se realizó por PCR en tiempo real. El análisis inmunohistoquímico mostró escaso etiquetado nuclear positivo para la proteína PARP y AIF en el cuerpo cavernoso de los animales de control y tratados con etanol. Después del análisis de la expresión de microARN miR-145, -210 y -486 no se encontraron resultados con diferencias estadísticas significativas entre los grupos control y alcoholizados. La expresión de AIF y PARP y sus microARN reguladores involucrados en el proceso apoptótico (miR-145, miR-210 y miR-486) no se alteraron en el cuerpo cavernoso de las ratas sometidas a alcoholismo semivoluntario.

Morphological and molecular analysis of apoptosis in the corpus cavernosum of rats submitted to a chronic alcoholism model.

PURPOSE: To evaluate the effect of chronic alcoholism on morphometry and apoptosis mechanism and correlate with miRNA-21 expression in the corpus cavernosum of rats. METHODS: Twenty-four rats were divided into two experimental groups: Control (C) and Alcoholic group (A). After two weeks of an adaptive phase, rats from group A received only ethanol solution (20%) during 7 weeks. The morphometric and caspase-3 immunohistochemistry analysis were performed in the corpus cavernosum. The miRNA-21 expression was analyzed in blood and cavernous tissue. RESULTS: Chronic ethanol consumption decreased cavernosal smooth muscle area of alcoholic rats. The protein expression of caspase 3 in the corpus cavernosum was higher in A compared to the C group. There was no difference in the expression of miRNA-21 in serum and cavernous tissue between the groups. CONCLUSION: Chronic ethanol consumption reduced smooth muscle area and increased caspase 3 in the corpus cavernosum of rats, without altered serum and cavernosal miR-21 gene expression.

Sensory and Motor Conduction Velocity in Spontaneously Hypertensive Rats: Sex and Aging Investigation.

The literature is extensive on how hypertension affects the morphology and function of the central nervous system (CNS) and is being focused on multiple organ damage involving the kidneys, heart, endothelium and retina. Hypertension damage to the peripheral nervous system is less explored in the literature. We have previously shown morphometric alterations in large and small caliber myelinated fibers of nerves in the adult spontaneously hypertensive rat (SHR). However, the functional correlation of these findings has not been explored. We performed an electrophysiological investigation of hind limb nerves in SHR of both genders in different ages. Normotensive Wistar-Kyoto (WKY) rats were used as controls. Electrophysiological recordings and determination of motor (MCV) and sensory (SCV) nerve conduction velocity were performed in the same animals at four different ages: 5, 8, 20 and 40 weeks after birth. Comparisons were made between ages, genders and animal strain. We showed a continuous body weight increase in adult life in all animals studied. MCV got stable at 20-week old hypertensive animals and continued to increase in normotensive ones. The SCV was constant between the ages of 20 and 40 weeks old in female SHR and decreased in male SHR while it continued to increase in WKY animals. The electrophysiological investigation of the nerves in WKY and SHR from both genders and different ages, associated with morphological and morphometric data from the literature suggest that hypertension affects the nerve function and might corroborate the development of a peripheral neuropathy.

Growth factors expression and ultrastructural morphology after application of low-level laser and natural latex protein on a sciatic nerve crush-type injury.

The effects of low-level laser therapy (LLLT) and natural latex protein (F1, Hevea brasiliensis) were evaluated on crush-type injuries (15kg) to the sciatic nerve in the expressions of nerve growth factor (NGF) and vascular endothelium growth factor (VEGF) and ultrastructural morphology to associate with previous morphometric data using the same protocol of injury and treatment. Thirty-six male rats were allocated into six experimental groups (n = 6): 1-Control; 2-Exposed nerve; 3-Injured nerve; 4-LLLT (15J/cm2, 780nm, 30mW, Continuous Wave) treated injured nerve; 5-F1 (0,1mg) treated injured nerve; and 6-LLLT&F1 treated injured nerve. Four or eight weeks after, sciatic nerve samples were processed for analysis. NGF expression were higher (p<0.05) four weeks after in all injured groups in comparison to Control (Med:0.8; Q1:0; Q3:55.5%area). Among them, the Injured (Med:70.7; Q1:64.4; Q3:77.5%area) showed the highest expression, and F1 (Med:17.3; Q1:14.1; Q3:21.7%area) had the lowest. At week 8, NGF expressions decreased in the injured groups. VEGF was expressed in all groups; its higher expression was observed in the injured groups 4 weeks after (Injured. Med:29.5; F1. Med:17.7 and LLLT&F1. Med:19.4%area). At week 8, a general reduction of VEGF expression was noted, remaining higher in F1 (Med:35.1; Q1.30.6; Q3.39.6%area) and LLLT&F1 (Med:18.5; Q1:16; Q3:25%area). Ultrastructural morphology revealed improvements in the treated groups; 4 weeks after, the F1 group presented greater quantity and diameter of the nerve fibers uniformly distributed. Eight weeks after, the F1 and LLLT&F1 showed similar characteristics to the non-injured groups. In summary, these results and our previous studies indicated that F1 and LLLT may favorably influence the healing of nerve crush injury. Four weeks after nerve injury F1 group showed the best results suggesting recovery acceleration; at 8th week F1 and LLLT&F1 groups presented better features and higher vascularization that could be associated with VEGF maintenance.

Substance P in Dorsal Root Ganglion Neurons in Young and Adult Rats, after Nociceptive Stimulation during the Neonatal Period.

The nervous system is highly plastic during the neonatal period, being sensitive to noxious stimuli, which may cause short- and long-term pain responsivity changes. Understanding plasticity in peripheral pain pathways is crucial, particularly when the nervous system is still under development and remodeling process. Substance P (SP) is widely used as a marker for peripheral neurons with unmyelinated and small myelinated fibers. We investigated the number of SP immunoreactive neurons in the dorsal root ganglion (DRG) of male and female Wistar rats, 15 and 180 days after nociceptive stimulation during the neonatal period. Right and left 5th lumbar (L5) DRG were incubated in rabbit polyclonal anti-substance P primary followed by biotinylated donkey anti-rabbit secondary antibodies. Reaction was revealed with a nickel-diaminobenzidine solution. Labeled neurons were counted and compared between ages, genders and groups. Gender differences were present in both ages, with the number of SP-positive DRG neurons being larger in 15-days-old males on both sides. After 180 days, males showed a larger number of SP-positive neurons than females only on the nociceptive stimulated side. An increased number of SP-positive neurons in the DRG on the stimulated side was present in females, immediately after nociceptive stimulation, but not after 180 days. In conclusion, neonatal noxious stimulation caused a permanent increase in SP-positive DRG neurons in males that was not observed in females, suggesting that differences in pain processing/responsivity between genders could be related to morphological alterations of the nervous system. Anat Rec, 301:849-861, 2018. © 2017 Wiley Periodicals, Inc.

Analysis of the variation in low-level laser energy density on the crushed sciatic nerves of rats: a morphological, quantitative, and morphometric study.

The objective of this study was to evaluate three energy densities of low-level laser therapy (LLLT, GaAlAs, 780 nm, 40 mW, 0.04 cm2) for the treatment of lesions to peripheral nerves using the sciatic nerve of rats injured via crushing model (15 kgf, 5.2 MPa). Thirty Wistar rats (♂, 200-250 g) were divided into five groups (n = 6): C-control, not injured, and irradiated; L0-injured nerve without irradiation; L4-injured nerve irradiated with LLLT 4 J/cm2 (0.16 J); L10-injured nerve irradiated with LLLT 10 J/cm2 (0.4 J); and L50-injured nerve irradiated with LLLT 50 J/cm2 (2 J). The animals were sacrificed 2 weeks after the injury via perfusion with glutaraldehyde (2.5%, 0.1 M sodium cacodylate buffer). The nerve tissue was embedded in historesin, cut (3 µm), mounted on slides, and stained (Sudan black and neutral red). The morphological and quantitative analysis (myelin and blood capillary densities) and morphometric parameters (maximum and minimum diameters of nerve fibers, axon diameter, G-ratio, myelin sheath thickness) were assessed using the ImageJ software. ANOVA (parametric) or Kruskal-Wallis (nonparametric) tests were used for the statistical analysis. Groups L0, L4, L10, and L50 exhibited diminished values of all the quantitative and morphometric parameters in comparison to the control group. The morphological, quantitative, and morphometric data revealed improvement after injury in groups L4, L10, and L50 (irradiated groups) compared to the injured-only group (L0); the best results, in general, were observed for the L10 group after 15 days of nerve injury.

Aspects of the macro and microscopic anatomy of the sciatic nerve in Wistar rats / Aspectos de la anatomía macro y microscópica del nervio ciático en ratas Wistar

The present study seeks to systematize morphological and morphometrical parameters and brings new data on the main branch of the lumbosacral plexus ­ i.e., sciatic nerve ­ in Wistar rats aged four and seven weeks. Sixteen female were divided into two groups, namely animals aged four weeks, and animals aged seven weeks. The specimens were studied at proximal and distal segments of the right hind limb sciatic nerves. Semi-thin transverse sections (0.25 µm thickness) were stained with 1 % toluidine blue, and the morphometric analysis was processed through the KS 400 software. Except for the number of fascicles and fascicular diameter, no differences were found between the proximal and distal segments. We observed differences when morphometric values were compared between 4- and 7- week old animals, with some exceptions (number of fascicles and myelinated fibers, and capillary area and number). The macroscopic data disagree with a previous description of the sciatic nerve being composed by two fascicles. Instead, sciatic nerve's only fascicle trifurcates or quadrifurcates at the distal third of the thigh. The total capillary area and density were calculated, and these are the first referential data for the sciatic nerve. Histograms of myelinated fiber and axons considering the animal ages were built. The results presented here are important because experimental studies, mainly studies on nerve regenerations require comparison with normal reliable data.

El objetivo fue sistematizar los parámetros morfológicos y morfométricos y traer nuevos datos sobre el ramo principal del plexo lumbosacro - es decir nervio ciático - en ratas Wistar de 4 a 7 semanas. Dieciséis ratas fueron divididas en dos grupos, con 4 y 7 semanas de edad. Las muestras estudiadas fueron los segmentos proximal y distal del nervio ciático derecho. Secciones delgadas (espesor 0,25 mm) fueron teñidas con azul de toluidina al 1 % y el análisis morfométrico se llevó a cabo utilizando el programa KS 400. Excepto para el número de fascículos y diámetro fascicular, no se encontraron diferencias entre los segmentos proximal y distal. Fueron observadas diferencias cuando se compararon los valores morfométricos entre animales de 4 y 7 semanas, con algunas excepciones (número de fascículos y fibras mielinizadas, área y número de capilares). Los datos macroscópicos no están de acuerdo con la descripción anterior del nervio ciático siendo compuesto por dos fascículos. En cambio, sólo trifurcación o cuadrifurcación fueron encontrados en el tercio distal del muslo. El área total capilar y la densidad fueron calculadas y estos constituyen los primeros datos de referencia para el nervio ciático. Se construyeron histogramas de fibras mielínicas y axones, teniendo en cuenta las edades de los animales. Los resultados presentados aquí son importantes porque los estudios experimentales, en especial aquellos sobre la regeneración nerviosa, requieren comparación con datos confiables normales.

Does Pain in the Neonatal Period Influence Motor and Sensory Functions in a Similar Way for Males and Females During Post-Natal Development in Rats?

OBJECTIVE : Early pain experiences can lead to disruption in the long-term responses to pain and in abnormal development and behavior in rodents. We evaluated the sensory and motor development of Wistar rats after exposure to painful stimulation (repetitive needle prickling) immediately after birth. METHODS : Male and female rats were followed up to 6 months of life, and sensory and motor functions were investigated by testing paw withdrawal with von Frey filaments, calibrated forceps (CF), and grip strength (GS) tests. RESULTS : Body weight increased with age and tended to be smaller in pain groups compared with their controls of the same sex. GS values also increased with age in controls but were stable and even decreased in pain groups from 120 up to 180 days. The von Frey filaments test showed higher values on the nonstimulated paws in male and female pain groups, with no differences between sides on the controls. The CF test showed smaller values on the stimulated paws in the pain group, with no differences between sides on the controls. CONCLUSIONS : Pain in the neonatal period influences sensory and motor functions negatively during development in male and female rats, even long term after the painful stimulus is ceased. The neonatal injury-induced hypersensitivity is persistent, and male and female rats respond similarly to the stimulus.

Recurrent laryngeal nerve alterations in developing spontaneously hypertensive rats.

OBJECTIVES/HYPOTHESIS: It is well known that the recurrent laryngeal nerve not only innervates the larynx but also contains baroreceptor fibers, as demonstrated by physiological studies. Because hypertension has a negative impact on both peripheral nerve morphology and the baroreflex, we investigated the recurrent laryngeal nerve morphological alterations related to the development of hypertension. METHODS: We compared morphological and morphometric aspects of different segments of the recurrent laryngeal nerve in male and female spontaneously hypertensive rats in different ages: 5, 8, and 20 weeks (n = 6 per group). Blood pressure and heart rate were recorded in anesthetized animals, followed by removal of the right and left recurrent laryngeal nerves for epoxy resin embedding and light microscopy analysis. Computer software was used for morphometric analysis. RESULTS: The blood pressure was significantly higher in 20-week-old animals compared to those at 5 weeks. Body weight increased significantly with age, as did the nerve fascicles. For the myelinated fibers and respective axons, there was a reduction of fiber size, more evident on the axon, associated with a reduction of the small myelinated fibers percentage in animals with high blood pressure. Also, 20-week-old animals showed a significant reduction of the blood vessel percentage of occupancy compared to younger ages. No differences were observed between genders. CONCLUSION: Hypertension development impaired axon growth, affecting mainly the small myelinated fibers. Males and females were affected equally. The alterations of the endoneural blood vessels probably played an important role on the small fibers alterations.

A New Method for Automated Identification and Morphometry of Myelinated Fibers Through Light Microscopy Image Analysis.

Nerve morphometry is known to produce relevant information for the evaluation of several phenomena, such as nerve repair, regeneration, implant, transplant, aging, and different human neuropathies. Manual morphometry is laborious, tedious, time consuming, and subject to many sources of error. Therefore, in this paper, we propose a new method for the automated morphometry of myelinated fibers in cross-section light microscopy images. Images from the recurrent laryngeal nerve of adult rats and the vestibulocochlear nerve of adult guinea pigs were used herein. The proposed pipeline for fiber segmentation is based on the techniques of competitive clustering and concavity analysis. The evaluation of the proposed method for segmentation of images was done by comparing the automatic segmentation with the manual segmentation. To further evaluate the proposed method considering morphometric features extracted from the segmented images, the distributions of these features were tested for statistical significant difference. The method achieved a high overall sensitivity and very low false-positive rates per image. We detect no statistical difference between the distribution of the features extracted from the manual and the pipeline segmentations. The method presented a good overall performance, showing widespread potential in experimental and clinical settings allowing large-scale image analysis and, thus, leading to more reliable results.

Hind limb sensory innervation in rats: comparison between sural and saphenous nerve morphometry / Inervación sensorial de los miembros posteriores en ratas: comparación morfométrica entre los nervios sural y safeno

Although numerous studies investigate sensory recovery of the hind paw of the rat after nerve damage, still no comprehensive overview of its normal innervation is present in the literature. We investigated the morphometry of myelinated fibers in the sural and saphenous nerves and analyzed their size distributions in young rats. Six 30-day-old female Wistar rats were perfused with 2.5% glutaraldehyde and their right and left sural and saphenous nerves were prepared for light microscopy and morphometry. Morphometric data were compared between segments (proximal versus distal) and sides (right versus left) for the same nerves. Also, segments from right or left sides were compared between nerves (sural versus saphenous). Both, the sural and saphenous nerves, exhibited proximal to distal symmetry on both sides as well as left-right symmetry. Histograms of the diameter of the myelinated fibers were unimodal in both nerves, regardless of segments or sides with the peaks of the fibers size occurring between 2.5 and 4.0 µm. The axon distributions reflected the myelinated fiber distributions, with the sural and saphenous nerves peaking between 1.5 and 2.0 µm. The G ratio (the ratio between the axon and fiber diameters) distributions were also unimodal, with peaks at 0.6 for both nerves. This study contributes to the literature with information on the myelinated fibers morphometry from the two sensory nerves responsible for the rat hind limb innervation. This information is valuable for a better understanding of the possible contribution of collateral sprouting from the sural or saphenous nerves on the paw sensory territory recovery observed after one of these nerves is damaged.

Aunque numerosos estudios investigan la recuperación sensorial del miembro pélvico o posterior de la rata después del daño en los nervios, aún no existe en la literatura una visión global de su inervación normal. Investigamos la morfometría de fibras mielínicas de los nervios sural y safeno y analizamos sus distribuciones de tamaño en ratas jóvenes. Seis ratas Wistar de 30 días de edad fueron perfundidas con 2,5% de glutaraldehído, se prepararon los nervios sural y safeno derecho e izquierdo para microscopía de luz y morfometría. Datos morfométricos fueron comparados entre los segmentos (proximal vs distal) y laterales (derecho vs izquierdo) para los mismos nervios. Además, los segmentos de los lados derecho e izquierdo se compararon entre los nervios (sural vs safeno). Ambos nervios sural y safeno exhibieron una simetría proximal a distal en ambos lados, así como una simetría izquierda-derecha. Histogramas del diámetro de las fibras mielinizadas eran unimodales en ambos nervios, independientemente de los segmentos o de los lados, siendo los peaks del tamaño de las fibras entre 2,5 y 4,0 micras. Las distribuciones de los axones reflejan las distribuciones de fibras mielinizadas, de los nervios sural y safeno que alcanzaban entre 1,5 and 2,0 µm. La relación de G (relación entre los diámetros de los axones y de fibra) eran también unimodales, alcanzando 0,6 para ambos nervios. Este estudio contribuye a la literatura con los datos de la morfometría de fibras mielinizadas de ambos nervios sensoriales responsables de la inervación de la extremidad pélvica de la rata. Esta información es valiosa para una mejor comprensión de los nervios sural y safeno en la recuperación sensorial del miembro después de que uno de estos nervios ha sido dañado.

Properties of the tibialis anterior muscle after treatment with laser therapy and natural latex protein following sciatic nerve crush.

INTRODUCTION: In this study we evaluated the characteristics of the tibialis anterior muscle after sciatic nerve crush and treatment with low-level laser therapy (LLLT) or the protein from natural latex (P1). METHODS: We studied the following 6 groups of male Wistar rats: control (CG); exposed nerve (EG); injured nerve (IG); injured nerve with LLLT (LG); injured nerve with P1 (PG); and injured nerve with P1 and LLLT (LPG). RESULTS: After 4 weeks, muscle morphology showed improvement in the treated groups; after 8 weeks, the treated groups resembled controls, especially the PG. Morphometry revealed muscle fiber atrophy after nerve injury, with time-dependent recovery. Histochemical analysis revealed increased intermediate fiber area. The PG was more similar to controls with NADH staining, whereas the LPG more closely resembled controls with SDH staining. CONCLUSION: Treatment using only P1 proved most efficient, revealing a negative interaction between P1 and LLLT.

Association of chronic diabetes and hypertension in sural nerve morphometry: an experimental study.

BACKGROUND: Prospective studies have shown incidence rates of hypertension in diabetes mellitus to be three times that of subjects without diabetes mellitus. The reverse also applies, with the incidence of diabetes two to three times higher in patients with hypertension. Despite this common clinical association, the contribution of each isolated entity in the development of a neuropathy is still not well understood. The aims of the present study were to investigate the presence of peripheral neuropathy in spontaneously hypertensive rats (SHR) and SHR with chronically induced diabetes, using a morphological and morphometric study of the sural nerves. METHODS: Female SHR and normotensive Wistar rats (WR), 8 weeks old, received a single intravenous injection of streptozotocin (STZ) through the tail vein. Controls from both strains received vehicle. Twelve weeks after the injection, sural nerves were dissected and prepared for light microscopy. Morphometry of sural nerve fascicles and myelinated fibers was performed with the aid of computer software. RESULTS: The sural nerve myelinated fibers were highly affected by experimental diabetes in normotensive rats, causing mainly the reduction of the fiber size. Hypertensive rats showed characteristics of small fiber neuropathy and a severe reduction of the number and density or Schwann cells. The association between diabetes and hypertension caused an increase on the average size of the myelinated fibers, pointing to a small fiber loss, associated to axonal atrophy. CONCLUSIONS: Our study gives morphological support to the existence of a neuropathy due to hypertension, which is among one of the most common risk factors for diabetic neuropathy. The association between the two neuropathies showed to be a complex alteration, involving and including both, large and small fibers neuropathy. Hypertension caused, indeed, an exacerbation of the alterations already observed in experimental models of diabetic neuropathy.