Transplantation of muscle-derived stem cells plus biodegradable fibrin glue restores the urethral sphincter in a pudendal nerve-transected rat model

We investigated whether fibrin glue (FG) could promote urethral sphincter restoration in muscle-derived stem cell (MDSC)-based injection therapies in a pudendal nerve-transected (PNT) rat, which was used as a stress urinary incontinence (SUI) model. MDSCs were purified from the gastrocnemius muscles of 4-week-old inbred female SPF Wistar rats and labeled with green fluorescent protein. Animals were divided into five groups (N = 15): sham (S), PNT (D), PNT+FG injection (F), PNT+MDSC injection (M), and PNT+MDSC+FG injection (FM). Each group was subdivided into 1- and 4-week groups. One and 4 weeks after injection into the proximal urethra, leak point pressure (LPP) was measured to assess urethral resistance function. Histology and immunohistochemistry were performed 4 weeks after injection. LPP was increased significantly in FM and M animals after implantation compared to group D (P < 0.01), but was not different from group S. LPP was slightly higher in the FM group than in the M group but there was no significant difference between them at different times. Histological and immunohistochemical examination demonstrated increased numbers of surviving MDSCs (109 ± 19 vs 82 ± 11/hpf, P = 0.026), increased muscle/collagen ratio (0.40 ± 0.02 vs 0.34 ± 0.02, P = 0.044), as well as increased microvessel density (16.9 ± 0.6 vs 14.1 ± 0.4/hpf, P = 0.001) at the injection sites in FM compared to M animals. Fibrin glue may potentially improve the action of transplanted MDSCs to restore the histology and function of the urethral sphincter in a SUI rat model. Injection of MDSCs with fibrin glue may provide a novel cellular therapy method for SUI.

Apoptosis of sensory neurons and satellite cells after sciatic nerve transection in C57BL/6J mice

The rate of axonal regeneration, after sciatic nerve lesion in adult C57BL/6J mice, is reduced when compared to other isogenic strains. It was observed that such low regeneration was not due just to a delay, since neuronal death was observed. Two general mechanisms of cell death, apoptosis and necrosis, may be involved. By using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) technique, we demonstrated that a large number of sensory neurons, as well as satellite cells found in the dorsal root ganglia, were intensely labeled, thus indicating that apoptotic mechanisms were involved in the death process. Although almost no labeled neurons or satellite cells were observed one week after transection, a more than ten-fold increase in TUNEL labeling was detected after two weeks. The results obtained with the C57BL/6J strain were compared with those of the A/J strain, which has a much higher peripheral nerve regeneration potential. In A/J mice, almost no labeling of sensory neurons or satellite cells was observed after one or two weeks, indicating the absence of neuronal loss. Our data confirm previous observations that approximately 40% of C57BL/6J sensory neurons die after sciatic nerve transection, and indicate that apoptotic events are involved. Also, our observations reinforce the hypothesis that the low rate of axonal regeneration occurring in C57BL/6J mice may be the result of a mismatch in the timing of the neurons need for neurotrophic substances, and production of the latter by non-neuronal cells in the distal stump.

Effects of systemic administration of ciliary neurotrophic factor on Bax and Bcl-2 proteins in the lumbar spinal cord of neonatal rats after sciatic nerve transection

Ciliary neurotrophic factor (CNTF) is a cytokine that plays a neuroprotective role in relation to axotomized motoneurons. We determined the effect of daily subcutaneous doses of CNTF (1.2 µg/g for 5 days; N = 13) or PBS (N = 13) on the levels of mRNA for Bcl-2 and Bax, as well as the expression and inter-association of Bcl-2 and Bax proteins, and the survival of motoneurons in the spinal cord lumbar enlargement of 2-day-old Wistar rats after sciatic nerve transection. Five days after transection, the effects were evaluated on histological and molecular levels using Nissl staining, immunoprecipitation, Western blot analysis, and reverse transcriptase-polymerase chain reaction. The motoneuron survival ratio, defined as the ratio between the number of motoneurons counted on the lesioned side vs those on the unlesioned side, was calculated. This ratio was 0.77 ± 0.02 for CNTF-treated rats vs 0.53 ± 0.02 for the PBS-treated controls (P < 0.001). Treatment with CNTF modified the level of mRNA, with the expression of Bax RNA decreasing 18% (with a consequent decrease in the level of Bax protein), while the expression of Bcl-2 RNA was increased 87%, although the level of Bcl-2 protein was unchanged. The amount of Bcl-2/Bax heterodimer increased 91% over that found in the PBS-treated controls. These data show, for the first time, that the neuroprotective effect of CNTF on neonatal rat axotomized motoneurons is associated with a reduction in free Bax, due to the inhibition of Bax expression, as well as increased Bcl-2/Bax heterodimerization. Thus, the neuroprotective action of the CNTF on axotomized motoneurons can be related to the inhibition of this apoptotic pathway.

Participation of neuronal nitric oxide synthase in experimental neuropathic pain induced by sciatic nerve transection

Nerve injury leads to a neuropathic pain state that results from central sensitization. This phenomenom is mediated by NMDA receptors and may involve the production of nitric oxide (NO). In this study, we investigated the expression of the neuronal isoform of NO synthase (nNOS) in the spinal cord of 3-month-old male, Wistar rats after sciatic nerve transection (SNT). Our attention was focused on the dorsal part of L3-L5 segments receiving sensory inputs from the sciatic nerve. SNT resulted in the development of neuropathic pain symptoms confirmed by evaluating mechanical hyperalgesia (Randall and Selitto test) and allodynia (von Frey hair test). Control animals did not present any alteration (sham-animals). The selective inhibitor of nNOS, 7-nitroindazole (0.2 and 2 µg in 50 µL), blocked hyperalgesia and allodynia induced by SNT. Immunohistochemical analysis showed that nNOS was increased (48% by day 30) in the lumbar spinal cord after SNT. This increase was observed near the central canal (Rexed’s lamina X) and also in lamina I-IV of the dorsal horn. Real-time PCR results indicated an increase of nNOS mRNA detected from 1 to 30 days after SNT, with the highest increase observed 1 day after injury (1469%). Immunoblotting confirmed the increase of nNOS in the spinal cord between 1 and 15 days post-lesion (20%), reaching the greatest increase (60%) 30 days after surgery. The present findings demonstrate an increase of nNOS after peripheral nerve injury that may contribute to the increase of NO production observed after peripheral neuropathy.

Effects of sciatic nerve transection on ultrastructure, NADPH-diaphorase reaction and serotonin-, tyrosine hydroxylase-, c-Fos-, glucose transporter 1- and 3-like immunoreactivities in frog dorsal root ganglion

Frogs have been used as an alternative model to study pain mechanisms. Since we did not find any reports on the effects of sciatic nerve transection (SNT) on the ultrastructure and pattern of metabolic substances in frog dorsal root ganglion (DRG) cells, in the present study, 18 adult male frogs (Rana catesbeiana) were divided into three experimental groups: naive (frogs not subjected to surgical manipulation), sham (frogs in which all surgical procedures to expose the sciatic nerve were used except transection of the nerve), and SNT (frogs in which the sciatic nerve was exposed and transected). After 3 days, the bilateral DRG of the sciatic nerve was collected and used for transmission electron microscopy. Immunohistochemistry was used to detect reactivity for glucose transporter (Glut) types 1 and 3, tyrosine hydroxylase, serotonin and c-Fos, as well as nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase). SNT induced more mitochondria with vacuolation in neurons, satellite glial cells (SGCs) with more cytoplasmic extensions emerging from cell bodies, as well as more ribosomes, rough endoplasmic reticulum, intermediate filaments and mitochondria. c-Fos immunoreactivity was found in neuronal nuclei. More neurons and SGCs surrounded by tyrosine hydroxylase-like immunoreactivity were found. No change occurred in serotonin- and Glut1- and Glut3-like immunoreactivity. NADPH-diaphorase occurred in more neurons and SGCs. No sign of SGC proliferation was observed. Since the changes of frog DRG in response to nerve injury are similar to those of mammals, frogs should be a valid experimental model for the study of the effects of SNT, a condition that still has many unanswered questions.

Pharmacological immunomodulation enhances peripheral nerve regeneration

To assess the effect of N-Acetylmuramyl-L-Alanyl-D-Isoglutamine MDP topically administrated on the regenerating peripheral neurons, twelve male C57BL/6J adult mice were equally distributed into three groups. Four mice underwent unilateral sciatic nerve transection and polyethylene tubulization, with a 4mm gap between the proximal and distal nerve stumps and were implanted with collagen + PBS (COL). Other four animals underwent the same surgical procedure but received collagen + MDP (COL/MDP) inside the prosthesis. Four animals were not operated and served as control group (NOR). After 4 weeks, the regenerated nerve cables were processed for total myelinated axon counting and myelinated fiber diameter measurement. The L5 dorsal root ganglion (DRG) was also removed and sectioned for sensory neurons counting and measurement. The results revealed significant difference (p<0.05) in axonal counting among the groups NOR (4,355±32), COL (1,869±289) and COL/MDP (2,430±223). There was a significant reduction in the axonal diameter in the operated groups (COL=3.38µm±1.16 and COL/MDP=3.54µm±1.16) compared to NOR (6.19µm±2.45). No difference was found in the number of DRG neurons between the experimental groups (COL=564±51; COL/MDP=514±56), which presented fewer sensory neurons compared to NOR (1,097±142). Data obtained indicate that locally applied MDP stimulates peripheral nerve regeneration in mice.

Substance P immunoreactivity in the lumbar spinal cord of the turtle Trachemys dorbigni following peripheral nerve injury

Immunoreactive substance P was investigated in turtle lumbar spinal cord after sciatic nerve transection. In control animals immunoreactive fibers were densest in synaptic field Ia, where the longest axons invaded synaptic field III. Positive neuronal bodies were identified in the lateral column of the dorsal horn and substance P immunoreactive varicosities were observed in the ventral horn, in close relationship with presumed motoneurons. Other varicosities appeared in the lateral and anterior funiculi. After axotomy, substance P immunoreactive fibers were reduced slightly on the side of the lesion, which was located in long fibers that invaded synaptic field III and in the varicosities of the lateral and anterior funiculus. The changes were observed at 7 days after axonal injury and persisted at 15, 30, 60 and 90 days after the lesion. These findings show that turtles should be considered as a model to study the role of substance P in peripheral axonal injury, since the distribution and temporal changes of substance P were similar to those found in mammals.

PPM1B and P-IKKβ expression levels correlated inversely with rat gastrocnemius atrophy after denervation

Activated inhibitor of nuclear factor-κB kinase β (IKKβ) is necessary and sufficient for denervated skeletal muscle atrophy. Although several studies have shown that Mg2+/Mn2+-dependent protein phosphatase 1B (PPM1B) inactivated IKKβ, few studies have investigated the role of PPM1B in denervated skeletal muscle. In this study, we aim to explore the expression and significance of PPM1B and phosphorylated IKKβ (P-IKKβ) during atrophy of the denervated gastrocnemius. Thirty young adult female Wistar rats were subjected to right sciatic nerve transection and were sacrificed at 0 (control), 2, 7, 14, and 28 days after denervation surgery. The gastrocnemius was removed from both the denervated and the contralateral limb. The muscle wet weight ratio was calculated as the ratio of the wet weight of the denervated gastrocnemius to that of the contralateral gastrocnemius. RT-PCR and Western blot analysis showed that mRNA and protein levels of PPM1B were significantly lower than those of the control group at different times after the initiation of denervation, while P-IKKβ showed the opposite trends. PPM1B protein expression persistently decreased while P-IKKβ expression persistently increased for 28 days after denervation. PPM1B expression correlated negatively with P-IKKβ expression by the Spearman test, whereas decreasing PPM1B expression correlated positively with the muscle wet weight ratio. The expression levels of PPM1B and P-IKKβ were closely associated with atrophy in skeletal denervated muscle. These results suggest that PPM1B and P-IKKβ could be markers in skeletal muscle atrophy.

Facial nerve palsy associated with leptospirosis

This case report describes the findings of a 27-year-old black male from Bahia, Brazil, who developed facial palsy during the convalescence phase of leptospirosis. The patient recovered without neurological sequel. This work calls attention to a possible association between leptospirosis and facial palsy.

Degree of disability, pain levels, muscle strength, and electromyographic function in patients with Hansen's disease with common peroneal nerve damage

INTRODUCTION: This study evaluated the degree of disability, pain levels, muscle strength, and electromyographic function (RMS) in individuals with leprosy. METHODS: We assessed 29 individuals with leprosy showing common peroneal nerve damage and grade 1 or 2 disability who were referred for physiotherapeutic treatment, as well as a control group of 19 healthy participants without leprosy. All subjects underwent analyses of degree of disability, electromyographic tests, voluntary muscle force, and the Visual Analog Pain Scale. RESULTS: McNemar's test found higher levels of grade 2 of disability (Δ = 75.9%; p = 0.0001) among individuals with leprosy. The Mann-Whitney test showed greater pain levels (Δ = 5.0; p = 0.0001) in patients with leprosy who had less extension strength in the right and left extensor hallucis longus muscles (Δ = 1.28, p = 0.0001; Δ = 1.55, p = 0.0001, respectively) and dorsiflexion of the right and left feet (Δ = 1.24, p = 0.0001; Δ = 1.45, p = 0.0001, respectively) than control subjects. The Kruskal-Wallis test showed that the RMS score for dorsiflexion of the right (Δ = 181.66 m·s-2, p = 0.001) and left (Δ = 102.57m·s-2, p = 0.002) feet was lower in patients with leprosy than in control subjects, but intragroup comparisons showed no difference. CONCLUSIONS: Leprosy had a negative influence on all of the study variables, indicating the need for immediate physiotherapeutic intervention in individuals with leprosy. This investigation opens perspectives for future studies that analyze leprosy treatment with physical therapeutic intervention.

Relationship between the recurrent laryngeal nerve and the inferior thyroid artery: a study in corpses

The anatomical relationship between the recurrent laryngeal nerve (RLN) and the inferior thyroid artery (ITA) was studied in 76 embalmed corpses, 8 females and 68 males. In both sexes, the RLN lay more frequently between branches of the ITA.; it was found in this position in 47.3% of male corpses and 42.8% of female ones. On the right, RLN was found between branches of the ITA in 49.3% of the cases, anterior to it in 38.04%, and posterior in 11.26%. On the left, the RLN lay between branches of the ITA in 44.45%, posterior to the ITA in 37.05%, and anterior to it in 18.05% of the cases. In 62.68% of the cases, the relationship found on one side did not occur again on the opposite side. There was a significant difference (p<0.05) in the distribution of the 3 types of relationships between the RLN and the ITA, on the right and on the left. Racial variations could contribute to an explanation of the differences observed by authors of different countries in the relationship between the RLN and the ITA.

Histopathological examination of nerve samples from pure neural leprosy patients: obtaining maximum information to improve diagnostic efficiency

Nerve biopsy examination is an important auxiliary procedure for diagnosing pure neural leprosy (PNL). When acid-fast bacilli (AFB) are not detected in the nerve sample, the value of other nonspecific histological alterations should be considered along with pertinent clinical, electroneuromyographical and laboratory data (the detection of Mycobacterium leprae DNA with polymerase chain reaction and the detection of serum anti-phenolic glycolipid 1 antibodies) to support a possible or probable PNL diagnosis. Three hundred forty nerve samples [144 from PNL patients and 196 from patients with non-leprosy peripheral neuropathies (NLN)] were examined. Both AFB-negative and AFB-positive PNL samples had more frequent histopathological alterations (epithelioid granulomas, mononuclear infiltrates, fibrosis, perineurial and subperineurial oedema and decreased numbers of myelinated fibres) than the NLN group. Multivariate analysis revealed that independently, mononuclear infiltrate and perineurial fibrosis were more common in the PNL group and were able to correctly classify AFB-negative PNL samples. These results indicate that even in the absence of AFB, these histopathological nerve alterations may justify a PNL diagnosis when observed in conjunction with pertinent clinical, epidemiological and laboratory data.

Cutaneous lesions sensory impairment recovery and nerve regeneration in leprosy patients

It is important to understand the mechanisms that enable peripheral neurons to regenerate after nerve injury in order to identify methods of improving this regeneration. Therefore, we studied nerve regeneration and sensory impairment recovery in the cutaneous lesions of leprosy patients (LPs) before and after treatment with multidrug therapy (MDT). The skin lesion sensory test results were compared to the histopathological and immunohistochemical protein gene product (PGP) 9.5 and the p75 nerve growth factor receptors (NGFr) findings. The cutaneous neural occupation ratio (CNOR) was evaluated for both neural markers. Thermal and pain sensations were the most frequently affected functions at the first visit and the most frequently recovered functions after MDT. The presence of a high cutaneous nerve damage index did not prevent the recovery of any type of sensory function. The CNOR was calculated for each biopsy, according to the presence of PGP and NGFr-immunostained fibres and it was not significantly different before or after the MDT. We observed a variable influence of MDT in the recovery from sensory impairment in the cutaneous lesions of LPs. Nociception and cold thermosensation were the most recovered sensations. The recovery of sensation in the skin lesions appeared to be associated with subsiding inflammation rather than with the regenerative activity of nerve fibres.

New sonographic measures of peripheral nerves: a tool for the diagnosis of peripheral nerve involvement in leprosy

To evaluate ultrasonographic (US) cross-sectional areas (CSAs) of peripheral nerves, indexes of the differences between CSAs at the same point (∆CSAs) and between tunnel (T) and pre-tunnel (PT) ulnar CSAs (∆TPTs) in leprosy patients (LPs) and healthy volunteers (HVs). Seventy-seven LPs and 49 HVs underwent bilateral US at PT and T ulnar points, as well as along the median (M) and common fibular (CF) nerves, to calculate the CSAs, ∆CSAs and ∆TPTs. The CSA values in HVs were lower than those in LPs (p < 0.0001) at the PT (5.67/9.78 mm2) and T (6.50/10.94 mm2) points, as well as at the M (5.85/8.48 mm2) and CF (8.17/14.14 mm2) nerves. The optimum CSA- receiver operating characteristic (ROC) points and sensitivities/specificities were, respectively, 6.85 mm2 and 68-85% for the PT point, 7.35 mm2 and 71-78% for the T point, 6.75 mm2 and 62-75% for the M nerve and 9.55 mm2 and 81-72% for the CF nerve. The ∆CSAs of the LPs were greater than those of the HVs at the PT point (4.02/0.85; p = 0.007), T point (3.71/0.98; p = 0.0005) and CF nerve (2.93/1.14; p = 0.015), with no difference found for the M nerve (1.41/0.95; p = 0.17). The optimum ∆CSA-ROC points, sensitivities, specificities and p-values were, respectively, 1.35, 49%, 80% and 0.003 at the PT point, 1.55, 55-85% and 0.0006 at the T point, 0.70, 58-50% and 0.73 for the M nerve and 1.25, 54-67% and 0.022 for the CF nerve. The ∆TPT in the LPs was greater than that in the HVs (4.43/1.44; p <0.0001). The optimum ∆TPT-ROC point was 2.65 (90% sensitivity/41% specificity, p < 0.0001). The ROC analysis of CSAs showed the highest specificity and sensitivity at the PT point and CF nerve, respectively. The PT and T ∆CSAs had high specificities (> 80%) and ∆TPT had the highest specificity (> 90%). New sonographic peripheral nerve measurements (∆CSAs and ∆TPT) provide an important methodological improvement in the detection of leprosy neuropathy.

Redundant nerve roots of the cauda equina: review of the literature

In imaging diagnosis, redundant nerve roots of the cauda equina are characterized by the presence of elongated, enlarged and tortuous nerve roots in close relationship with a high-grade lumbar spinal canal stenosis. This is not an independent entity, but it is believed to be a consequence of the chronic compression at the level of the lumbar canal stenosis and thus may be part of the natural history of lumbar spinal stenosis. The present paper is aimed at reviewing the histopathological, electrophysiological and imaging findings, particularly at magnetic resonance imaging, as well as the clinical meaning of this entity. As the current assessment of canal stenosis and root compression is preferably performed by means of magnetic resonance imaging, this is the imaging method by which the condition is identified. The recognition of redundant nerve roots at magnetic resonance imaging is important, particularly to avoid misdiagnosing other conditions such as intradural arteriovenous malformations. The literature approaching the clinical relevance of the presence of redundant nerve roots is controversial. There are articles suggesting that the pathological changes of the nerve roots are irreversible at the moment of diagnosis and therefore neurological symptoms are less likely to improve with surgical decompression, but such concept is not a consensus.