Luciana Sayuri Sanada

Microscopic anatomy of the sural nerve in the postnatal developing rat: a longitudinal and lateral symmetry study

Rat sural nerve is widely used in experimental studies investigating injury and regeneration of the peripheral nervous system. However, it has not yet been established whether morphological and morphometric parameters differ within corresponding levels of the rat sural nerve. The aims of the present study were to investigate the normal morphological and morphometric aspects of the sural nerve in postnatal developing female rats, with special attention to longitudinal morphology and lateral symmetry. Rats aged 30, 90 and 180 days were killed, and proximal and distal segments of the right and left sural nerves were prepared for light microscopy and morphometric study. No differences were found between the proximal and distal segments or between the right and left sides at the same levels. In addition, postnatal growth continuously and symmetrically affected the sural nerve fascicles and myelinated fibres. Fibre population distribution was also affected by increasing body weight; distribution was unimodal at 30 days, and by 180 days this distribution was established as bimodal. We concluded that the sural nerve is long and constant in its morphology and presents a continuous and symmetrical growth, more pronounced between 30 and 90 days of age, thus providing a good model for experimental neuropathies.

Spinal cord stimulation reduces mechanical hyperalgesia and glial cell activation in animals with neuropathic pain.

BACKGROUND:Spinal cord stimulation (SCS) is commonly used for neuropathic pain; the optimal variables and mechanisms of action are unclear. We tested whether modulation of SCS variables improved analgesia in animals with neuropathic pain by comparing 6-hour vs 30-minute duration and 50%, 75%, or 90% motor threshold (MT) intensity (amplitude). Furthermore, we examined whether maximally effective SCS reduced glial activation in the spinal cord in neuropathic animals. METHODS:Sprague-Dawley rats received the spared nerve injury model and were implanted with an epidural SCS lead. Animals were tested for mechanical withdrawal threshold of the paw before and 2 weeks after spared nerve injury, before and after SCS daily for 4 days, and 1, 4, and 9 days after SCS. Spinal cords were examined for the effects of SCS on glial cell activation. RESULTS:The mechanical withdrawal threshold decreased, and glial immunoreactivity increased 2 weeks after spared nerve injury. For duration, 6-hour SCS significantly increased the mechanical withdrawal threshold when compared with 30-minute SCS or sham SCS; 30-minute SCS was greater than sham SCS. For intensity (amplitude), 90% MT SCS significantly increased the withdrawal threshold when compared with 75% MT SCS, 50% MT SCS, and sham SCS. Both 4 and 60 Hz SCS decreased glial activation (GFAP, MCP-1, and OX-42) in the spinal cord dorsal horn when compared with sham. CONCLUSIONS:Six-hour duration SCS with 90% MT showed the largest increase in mechanical withdrawal threshold, suggesting that the variables of stimulation are important for clinical effectiveness. One potential mechanism for SCS may be to reduce glial activation at the level of the spinal cord.

Increasing Intensity of TENS Prevents Analgesic Tolerance in Rats

UNLABELLED Transcutaneous electrical nerve stimulation (TENS) reduces hyperalgesia and pain. Both low-frequency (LF) and high-frequency (HF) TENS, delivered at the same intensity (90% motor threshold [MT]) daily, result in analgesic tolerance with repeated use by the fifth day of treatment. The current study tested 1) whether increasing intensity by 10% per day prevents the development of tolerance to repeated TENS; and 2) whether lower intensity TENS (50% MT) produces an equivalent reduction in hyperalgesia when compared to 90% MT TENS. Sprague-Dawley rats with unilateral knee joint inflammation (3% carrageenan) were separated according to the intensity of TENS used: sham, 50% LF, 50% HF, 90% LF, 90% HF, and increased intensity by 10% per day (LF and HF). The reduced mechanical withdrawal threshold following the induction of inflammation was reversed by application of TENS applied at 90% MT intensity and increasing intensity for the first 4 days. On the fifth day, the groups that received 90% MT intensity showed tolerance. Nevertheless, the group that received an increased intensity on each day still showed a reversal of the mechanical withdrawal threshold with TENS. These results show that the development of tolerance can be delayed by increasing intensity of TENS. PERSPECTIVE Our results showed that increasing intensity in both frequencies of TENS was able to prevent analgesic tolerance. Results from this study suggest that increasing intensities could be a clinical method to prevent analgesic tolerance and contribute to the effective use of TENS in reducing inflammatory pain and future clinical trials.

Sural nerve involvement in experimental hypertension: morphology and morphometry in male and female normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR)

BackgroundThe sural nerve has been widely investigated in experimental models of neuropathies but information about its involvement in hypertension was not yet explored. The aim of the present study was to compare the morphological and morphometric aspects of different segments of the sural nerve in male and female spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. Rats aged 20 weeks (N = 6 in each group) were investigated. After arterial pressure and heart rate recordings in anesthetized animals, right and left sural nerves were removed and prepared for epoxy resin embedding and light microscopy. Morphometric analysis was performed with the aid of computer software, and took into consideration the fascicle area and diameter, as well as myelinated fiber number, density, area and diameter.ResultsSignificant differences were observed for the myelinated fiber number and density, comparing different genders of WKY and SHR. Also, significant differences for the morphological (thickening of the endoneural blood vessel walls and lumen reduction) and morphometric (myelinated fibers diameter and G ratio) parameters of myelinated fibers were identified. Morphological exam of the myelinated fibers suggested the presence of a neuropathy due to hypertension in both SHR genders.ConclusionsThese results indicate that hypertension altered important morphometric parameters related to nerve conduction of sural nerve in hypertensive animals. Moreover the comparison between males and females of WKY and SHR allows the conclusion that the morphological and morphometric parameters of sural nerve are not gender related. The morphometric approach confirmed the presence of neuropathy, mainly associated to the small myelinated fibers. In conclusion, the present study collected evidences that the high blood pressure in SHR is affecting the sural nerve myelinated fibers.

Spinal Cord Stimulation (SCS) Improves Decreased Physical Activity Induced by Nerve Injury

Spinal cord stimulation (SCS) is used to manage treatment of neuropathic pain to reduce pain and hyperalgesia and to improve activity. Prior studies using animal models of neuropathic pain have shown that SCS reduces hyperalgesia; however, it is unclear whether SCS affects physical activity. Therefore, we tested whether nerve injury (spared nerve injury [SNI] model) reduced physical activity levels, and whether SCS could restore these decreased activity levels. We tested whether SCS given over a long duration (6 hr daily for 3 months) remained effective. We compared SNI with uninjured controls over 4 weeks, and SNI with sham SCS with SNI with active SCS (4 or 60 Hz at 90% motor threshold). We confirmed the presence of mechanical hyperalgesia by examining mechanical thresholds of the paw with von Frey filaments. Physical activity levels were monitored over 30 min in an automated activity chamber as follows: overall activity, distance traveled, grooming behaviors, and rearing. Measures were taken during SCS every 1-2 weeks for 3 months. Animals with SNI (and no or sham SCS) showed decreased withdrawal thresholds ipsilaterally and reduced physical activity (rearing, distance, lines crossed) for 3 months. Both 4- and 60-Hz SCS increased paw withdrawal threshold during and immediately after SCS through 3 months. Both 4- and 60-Hz SCS increased the overall activity (lines crossed), distance traveled, and rearing, but not grooming behaviors for 3 months. This effect remained similar across the 3 months. Thus, measurement of spontaneous physical activity could be useful to examine nocifensive behaviors after nerve injury and is sensitive to SCS.

Cortex glial cells activation, associated with lowered mechanical thresholds and motor dysfunction, persists into adulthood after neonatal pain

We investigated if changes in glial activity in cortical areas that process nociceptive stimuli persisted in adult rats after neonatal injury. Neonatal pain was induced by repetitive needle prickling on the right paw, twice per day for 15 days starting at birth. Wistar rats received either neonatal pain or tactile stimulation and were tested behaviorally for mechanical withdrawal thresholds of the paws and gait alterations, after 15 (P15) or 180 (P180) days of life. Brains from rats on P15 and P180 were immunostained for glial markers (GFAP, MCP‐1, OX‐42) and the following cortical areas were analyzed for immunoreactivity density: prefrontal, anterior insular, anterior cingulated, somatosensory and motor cortices. Withdrawal thresholds of the stimulated paw remained decreased on P180 after neonatal pain when compared to controls. Neonatal pain animals showed increased density for both GFAP and MCP‐1 staining, but not for OX‐42, in all investigated cortical areas on both experimental times (P15 and P180). Painful stimuli in the neonatal period produced pain behaviors immediately after injury that persisted in adult life, and was accompanied by increase in the glial markers density in cortical areas that process and interpret pain. Thus, long‐lasting changes in cortical glial activity could be, at least in part, responsible for the persistent hyperalgesia in adult rats that suffered from neonatal pain.

Reproducibility in nerve morphometry: comparison between methods and among observers.

We investigated the reproducibility of a semiautomated method (computerized with manual intervention) for nerve morphometry (counting and measuring myelinated fibers) between three observers with different levels of expertise and experience with the method. Comparisons between automatic (fully computerized) and semiautomated morphometric methods performed by the same computer software using the same nerve images were also performed. Sural nerves of normal adult rats were used. Automatic and semiautomated morphometry of the myelinated fibers were made through the computer software KS-400. Semiautomated morphometry was conducted by three independent observers on the same images, using the semiautomated method. Automatic morphometry overestimated the myelin sheath area, thus overestimating the myelinated fiber size and underestimating the axon size. Fiber distributions overestimation was of 0.5 μm. For the semiautomated morphometry, no differences were found between observers for myelinated fiber and axon size distributions. Overestimation of the myelin sheath size of normal fibers by the fully automatic method might have an impact when morphometry is used for diagnostic purposes. We suggest that not only semiautomated morphometry results can be compared between different centers in clinical trials but it can also be performed by more than one investigator in one single experiment, being a reliable and reproducible method.

Can Wistar rats be used as the normotensive controls for nerve morphometry investigations in spontaneously hypertensive rats (SHR)

PURPOSE We compared the sural nerve morphology among Wistar (WR), Wistar-Kyoto (WKY) and Spontaneously hypertensive (SHR) rats, including the nerve fascicles and myelinated fibers morphometry. METHODS Age matched (20 weeks) female WR (N=6), WKY (N=6) and SHR (N=7) had their right and left sural nerves removed, embedded in epoxy resin, and observed by light microscopy. Morphometric analysis was performed with the aid of computer software. RESULTS Despite presenting the same age, WR were heavier than WKY and SHR, as were SHR compared to WKY. Systolic arterial pressure was higher in SHR compared to WR, but no differences between SHR and WKY or WR and WKY were observed. The sural nerves were morphometrically symmetric between proximal and distal segments on the same side and between sides in all strains with no differences in the myelinated fiber number. Schwann cell number and density were smaller in SHR and G ratio was larger in SHR, indicating that SHR have thinner myelinated fibers. CONCLUSION Sural nerve morphology is similar between WKY and WR, allowing the use of WR as the SHR controls in morphological investigations involving peripheral neuropathies.

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.

Hind Limb Sensory Innervation in Rats: Comparison between Sural and Saphenous Nerve Morphometry

Aunque numerosos estudios investigan la recuperacion sensorial del miembro pelvico o posterior de la rata despues del dano en los nervios, aun no existe en la literatura una vision global de su inervacion normal. Investigamos la morfometria de fibras mielinicas de los nervios sural y safeno y analizamos sus distribuciones de tamano en ratas jovenes. Seis ratas Wistar de 30 dias de edad fueron perfundidas con 2,5% de glutaraldehido, se prepararon los nervios sural y safeno derecho e izquierdo para microscopia de luz y morfometria. Datos morfometricos fueron comparados entre los segmentos (proximal vs distal) y laterales (derecho vs izquierdo) para los mismos nervios. Ademas, los segmentos de los lados derecho e izquierdo se compararon entre los nervios (sural vs safeno). Ambos nervios sural y safeno exhibieron una simetria proximal a distal en ambos lados, asi como una simetria izquierda-derecha. Histogramas del diametro de las fibras mielinizadas eran unimodales en ambos nervios, independientemente de los segmentos o de los lados, siendo los peaks del tamano 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 relacion de G (relacion entre los diametros de los axones y de fibra) eran tambien unimodales, alcanzando 0,6 para ambos nervios. Este estudio contribuye a la literatura con los datos de la morfometria de fibras mielinizadas de ambos nervios sensoriales responsables de la inervacion de la extremidad pelvica de la rata. Esta informacion es valiosa para una mejor comprension de los nervios sural y safeno en la recuperacion sensorial del miembro despues de que uno de estos nervios ha sido danado.