CTNNB1 Mutations and Estrogen Receptor Expression in Neuromuscular Choristoma and Its Associated Fibromatosis.

Neuromuscular choristoma (NMC) is a very rare, developmental malformation characterized by the endoneurial intercalation of mature muscle fibers among peripheral nerve fibers. NMC typically arises in the major proximal peripheral nerves, most commonly the sciatic nerve, and may involve the lumbosacral and brachial plexus. Patients present clinically with progressive neuropathy or plexopathy. NMC is strongly associated with development of a fibromatosis, histologically identical to conventional desmoid-type fibromatosis (NMC-fibromatosis). The development of NMC-fibromatosis is often precipitated by iatrogenic trauma (ie, biopsy). Desmoid-type fibromatosis is characterized by CTNNB1 exon 3 mutations, which result in aberrant nuclear ß-catenin localization and dysregulated canonical Wnt signaling. In contrast, the pathogenesis of NMC and NMC-fibromatosis is unknown. Desmoid-type fibromatosis expresses estrogen receptors (ER), specifically the ER-beta isoform (ERß), and endocrine therapies may be used in surgically unresectable cases. In contrast, the ER expression profile of NMC-fibromatosis is unknown. We evaluated a series of NMC and NMC-fibromatosis for CTNNB1 mutations, ß-catenin expression, and ER isoform expression. Five NMCs occurred in 2 female and 3 male patients (median age: 14 y, range <1 to 42 y), as masses involving the sciatic nerve (N=4) or brachial plexus (N=1). Four (of 5) NMCs had CTNNB1 mutations: 3 c.134 C>T (p.S45F) and 1 c.121 A>G (p.T41A). Four patients subsequently developed NMC-fibromatosis, and all 4 cases contained CTNNB1 mutations, including 1 p.T41A and 3 p.S45F mutations. In 3 patients, the NMC and NMC-fibromatosis had identical CTNNB1 mutations. Only 1 NMC had no detectable CTNNB1 mutation; however, the patient's subsequent NMC-fibromatosis had a CTNNB1 p.T41A mutation. All NMC and NMC-fibromatosis showed aberrant nuclear localization of ß-catenin, nuclear ERß expression, and no ERα expression. The presence of CTNNB1 mutations both in NMC and NMC-fibromatosis may be a shared molecular genetic abnormality underlying their pathogenesis.

Expression profiles of MiRNAs for intrinsic musculature of the forepaw and biceps in the rat model simulating irreversible muscular atrophy of obstetric brachial plexus palsy.

Clinically in obstetric brachial plexus palsy (OBPP), irreversible atrophy of intrinsic musculature of the hand in denervation occurs much earlier than that of denervated arm muscles. With the aim of finding clues to explain this, the miRNA expression profile of denervated intrinsic musculature of the forepaw (IMF) and that of the denervated biceps were examined by microarray screening in the rat model simulating irreversible muscular atrophy caused by pan-plexus lesions of OBPP, and potential targets of specifically dysregulated miRNAs were predicted with use of bioinformatics analysis. It was found that denervated IMF and biceps had their own specifically dysregulated miRNAs, respectively, as compared with control. Analysis of Gene Ontology and of pathway showed that those specifically dysregulated miRNAs and their target genes might participate in self-regulation of neuromuscular junctions (NMJs). These outcomes suggest that self-regulative mechanism of NMJs may be different between denervated IMF and denervated biceps in the rat model simulating irreversible muscular atrophy of OBPP.

Expression of activating transcription factor 3 (ATF3) in uninjured dorsal root ganglion neurons in a lower trunk avulsion pain model in rats.

PURPOSE: Clinically, neuropathic pain is frequent and intense following brachial plexus injury. It is thought that brachial plexus pain is not generated by avulsed roots, but rather by non-avulsed roots, since the avulsed root could not possibly transmit action potentials to central nerves. The aim of this study was to evaluate pain behavior and activation of sensory neurons in a brachial plexus avulsion (BPA) model in rats. METHODS: Fifteen male Wistar rats were used. In the BPA group, the C8-T1 roots were avulsed from the spinal cord with forceps at the lower trunk level (n = 5). In the naïve group, rats did not receive any procedures (n = 5). In the sham-operated group, the lower trunk was simply exposed (n = 5). Mechanical hyperalgesia of forelimbs corresponding to C6 and C7 dermatomes was measured using von Frey filaments every third day for 3 weeks. Activation of DRG neurons was immunohistochemically examined using anti-ATF3 (a marker for neuron activation) antibodies 21 days after surgery. Von Frey and immunohistochemical data between groups were analyzed using a Kruskal-Wallis test, followed by Mann-Whitney U tests. Bonferroni corrections were performed. RESULTS: Animals in the BPA group displayed significant mechanical hyperalgesia at the dermatome innervated by uninjured nerves continuing through day 21 compared with animals in the sham-operated group. ATF3-immunoreactive small and large DRG neurons were significantly activated in the BPA group (10.6 ± 9.5 and 5.2 ± 4.1 %, 39.7 ± 6.7 and 25.2 ± 10.3 %, 78.0 ± 9.1 and 53.7 ± 29.3 %) compared with the sham-operated group (0.7 ± 0.9 and 0 ± 0 %, 2.8 ± 2.0 and 1.0 ± 2.0 %, 3.9 ± 2.7 and 8.6 ± 10.1 %) at every level of C5, 6, and 7. In the naïve group, no DRG neurons were activated. ATF3-immunoreactive small and large DRG neurons were significantly activated at the level of C7 compared with C6 and C5, and significantly activated at the level of C6 compared with C5 in the BPA group. CONCLUSIONS: Expression of ATF3 in uninjured DRG neurons may contribute to pain following brachial plexus avulsion injury. Consequently, spared spinal sensory nerves may represent therapeutic targets for treatment of this pain.

Activation of astrocytes and microglia in the C3-T4 dorsal horn by lower trunk avulsion in a rat model of neuropathic pain.

PURPOSE: Brachial plexus pain is thought to be generated not by avulsed roots but rather by nonavulsed roots, because avulsed roots could not transmit action potentials to central nerves. The aim of this study was to evaluate pain-related behavior and the extent of glial activation in a model of brachial plexus avulsion (BPA). METHODS: We used 24 male Wistar rats. For rats in the BPA group, the C8-T1 roots were avulsed from the spinal cord at the level of the lower trunk (n = 10). Rats in a sham-surgery group had a similar surgery without the root avulsion (n = 7). Rats in an untreated group had no surgery (n = 7). Mechanical hyperalgesia of the forelimb plantar surfaces corresponding to C6 and C7 dermatomes was evaluated using a Semmes-Weinstein monofilament test every third day for 3 weeks (n = 15). Activation of astrocytes and microglia was examined immunohistochemically using anti-glia fibrillary acidic protein and anti-Iba1 antibodies 3 days after surgery (n = 9). RESULTS: When compared with rats in the sham-surgery and naive control groups, rats in the BPA group displayed significant mechanical hyperalgesia in the dermatome innervated by uninjured nerves both ipsilaterally and contralaterally and continuing through day 21. Iba1-immunoreactive microglia and glia fibrillary acidic protein-immunoreactive astrocytes were significantly activated on the ipsilateral side in the BPA group from levels C3 to T3 compared with the sham-surgery and untreated groups of rats. CONCLUSIONS: Activation of glia at uninjured levels of the dorsal horn may facilitate pain transmission following BPA injury. Consequently, spared spinal glial cells may represent therapeutic targets for treatment of pain related to BPA injury. CLINICAL RELEVANCE: Our findings may indicate why neuropathic pain is so frequent and intense following BPA injury.

The effect of Anti-NGF receptor (p75 Neurotrophin Receptor) antibodies on nociceptive behavior and activation of spinal microglia in the rat brachial plexus avulsion model.

STUDY DESIGN: We measured the response of the behavior and spinal glial activation to anti-nerve growth factor receptor (p75 neurotrophin receptor [p75NTR]) antibodies in the rat brachial plexus avulsion (BPA) model. OBJECTIVE: The aim of this study was to investigate the effect of anti-p75NTR antibodies on nociceptive behavior and activation of spinal microglia in the rat BPA model. SUMMARY OF BACKGROUND DATA: Tanezumab (anti-nerve growth factor antibody) treatment is associated with pain reduction and improvement in function, but with several complications. METHODS: Thirty male Wistar rats were used. In the BPA group, the C8-T1 roots were avulsed from the spinal cord with forceps at the lower trunk level and 10 µL of saline was applied locally (n = 10). In the anti-p75NTR group, the C8-T1 roots were avulsed and 10 µL of anti-p75NTR antibody was applied locally (n = 10). In a sham-operated group, the lower trunk was simply exposed (n = 10). Mechanical hyperalgesia and pain-induced walking patterns were measured using von Frey filaments (Stoelting, Wood Dale, IL) and the CatWalk gait analysis (Noldus Information Technology, the Netherlands) system every third day for 3 weeks. Activation of astrocytes and microglia was immunohistochemically examined in the spinal cord using anti-glial fibrillary acidic protein (GFAP) and anti-Iba1 antibodies both 7 and 21 days after surgery. RESULTS: Animals in the BPA group displayed significant mechanical hyperalgesia that continued through day 21 compared with animals in the sham-operated group, and mechanical hyperalgesia in the anti-p75NTR group was significantly improved 6 days after the operation. Regarding pain-induced gait analysis via CatWalk, animals in the BPA group displayed a significantly greater pain-like gait pattern than the p75 group for up to 3 weeks. Levels of GFAP-immunoreactive astrocytes and Iba1-immunoreactive microglia in the anti-p75NTR group were significantly reduced compared with the BPA group. CONCLUSION: Our results suggest that p75NTR contributes to neuropathic pain associated with BPA, and that inhibition of p75NTR reduces neuropathic pain. LEVEL OF EVIDENCE: N/A.

[Effect of the heat shock protein 27 on NOS expression of spinal cord anterior hornmotoneurons after brachial plexus avulsion].

AIM: To observe the effect of heat shock protein 27 (Hsp27) on nitric oxide synthase (NOS) of spinal cord anterior horn after brachial plexus roots avulsion. METHODS: Sixty male adult Wistar rats were divided into control and experiment groups at random. The experiment group subjected to heat shock under 45 degrees C for 15 min, and maintained under 42 degrees C for 20 min subsequently. After recovering 24 h under the room temperature, the nerves of brachial plexus were avulsion with microhemostatic forcep. In a span from 12 h to 7 d, these animals were killed at different time. But the control group only received the surgery of the nerve roots of brachial plexus avulsion. The freeze sections of spinal cord were stained by NADPH-d histochemistry, HSP27 immunohistochemical. RESULTS: (1) In experiment group, the motoneuron began to express NOS abundantly at 12 h after avulsion (A=0.13625). Then the NOS-positive neurons declined quickly, but in control group, the motoneuron began to express NOS at the 5th day after lesion. (2) Hsp27 begin to show the peak at 1 d in experiment and control groups, but the experiment group were more strong than the control group. CONCLUSION: Hsp27 inhibited NOS of motoneuron after avulsion and brought into full play the cytoprotection.

Expression of calcitonin gene-related peptide in anterior and posterior horns of the spinal cord after brachial plexus injury.

This study shows the expression pattern of calcitonin gene-related peptide (CGRP) in the anterior and posterior horns of the spinal cord after brachial plexus injury. The animals were divided into three injury groups: group 1, right C(7) anterior root avulsion; group 2, right C(7) anterior root avulsion and cut right C(5)-T(1) posterior roots; and group 3, right C(7) anterior root avulsion plus right hemitransection between the C(5) and C(6) segments of the spinal cord. These animals were killed at 1, 3, 7 and 14 days after injury. In the anterior horn of all three injured groups, the expression of CGRP increased progressively from day 1 to day 7 (p<0.05), peaked on day 7, and then began to decrease slowly. In the posterior horn of all three injured groups, the expression of CGRP decreased gradually from day 1 to day 14 after the operation and was significantly lower on day 14 compared to day 1. At each time point (days 1, 3, 7 and 14), the expression of CGRP was the highest in group 1 and the lowest in group 2, with significant differences among the three groups. The CGRP in the anterior horn of the spinal cord was derived from the cell bodies of motor neurons and was possibly involved in repair mechanisms and regeneration after nerve injury. However, the CGRP in the posterior horn was mainly derived from the posterior root ganglion and was possibly associated with the conduction of noxious stimulation.

SEPT9 mutations and a conserved 17q25 sequence in sporadic and hereditary brachial plexus neuropathy.

BACKGROUND: The clinical characteristics of sporadic brachial plexus neuropathy (S-BPN) and hereditary brachial plexus neuropathy (H-BPN) are similar. During attacks, inflammation of the brachial plexus nerves has been identified in both conditions. SEPT9 mutations (Arg88Trp, Ser93Phe, 5'UTR c.-131G>C) occur in some families with H-BPN. These mutations were not found in North American kindreds with H-BPN with a conserved 500-kilobase sequence of DNA at the 17q25 chromosomal region (where SEPT9 localizes) where a founder mutation has been suggested. OBJECTIVE: To study the 17q25 sequence and SEPT9 in S-BPN (56 individuals) and H-BPN (13 kindreds). METHODS: Allele analysis at 17q25, SEPT9 DNA sequencing, and messenger RNA analysis from lymphoblast cultures were performed. RESULTS: A conserved 17q25 sequence was found in 5 of 13 kindreds with H-BPN and 1 individual with S-BPN. This conserved sequence was not found in the family with a SEPT9 mutation (Arg88Trp) or in 182 control subjects. SEPT9 messenger RNA expression levels did not differ between forms of H-BPN and control subjects. No known mutations of SEPT9 were found in S-BPN. CONCLUSIONS: Rarely, individuals with S-BPN may have the same conserved 17q25 sequence found in many North American kindreds with H-BPN. Individuals with BPN with this conserved sequence do not seem to have SEPT9 mutations or alterations of messenger RNA expression levels in lymphoblast cultures and are predicted to have the most common genetic cause in North America by a founder-effect mutation.

Histopathological basis of Horner's syndrome in obstetric brachial plexus palsy differs from that in adult brachial plexus injury.

Although Horner's syndrome is usually taken as an absolute indicator of avulsions of the C8 and T1 ventral roots in adult brachial plexus injury, its pathological basis in obstetric brachial plexus palsy (OBPP) is unclear. We therefore examined the morphological mechanism for the presence of Horner's syndrome in brachial plexus injury in infants and adults. Some axons of sympathetic preganglionic neurons in T1 innervate the superior cervical ganglion via the C7 ventral root in infants but not in adults. Therefore, the presence of Horner's syndrome may relate in part to avulsion of the C7 root in OBPP. These findings suggest that Horner's syndrome in OBPP is not necessarily indicative of avulsions of the C8 and T1 roots, as it can occur with avulsion of the C7 root.

Some gamma-motoneurons contain gamma-aminobutyric acid in the rat cervical spinal cord.

Gamma-aminobutyric acid (GABA) is utilized in the peripheral as well as central nervous system. In this study, fibers immunoreactive for 67 kDa isoform of glutamic acid decarboxylase (GAD67), an enzyme which synthesizes GABA, were found to terminate in the intercapsular region of muscle spindles of the upper limb. GABA-containing fibers were also found in the ventral roots of C5 to T5 spinal segments, brachial plexus, and radial nerve. These fibers were thin and immunoreactive for choline-acetyl transferase (ChAT). After transection of the brachial plexus, GABA immunoreactivity disappeared completely in the ipsilateral triceps brachii muscle (TBM). After the injection of fluorogold into the TBM, some retrogradely labeled medium-sized neurons were positive for GAD67, but not VGAT mRNA. All these observations clearly indicate that GABA-containing gamma-motoneurons in the lower cervical spinal cord send their fibers to muscle spindles in the upper extremities. Since we detected neither GABAA nor GABAB receptors in the TBM by RT-PCR, the function of the GABA-containing gamma-motoneurons remains unclear.