Surgical correction of shoulder rotation deformity in brachial plexus birth palsy: long-term results in 118 patients.

We present the long-term results of open surgery for internal shoulder rotational deformity in brachial plexus birth palsy (BPBP). From 1997 to 2005, 207 patients (107 females, 100 males, mean age 6.2 (0.6 to 34)) were operated on with subscapularis elongation and/or latissimus dorsi to infraspinatus transfer. Incongruent shoulder joints were relocated. The early results of these patients has been reported previously. We analysed 118 (64 females, 54 males, mean age 15.1 (7.6 to 34)) of the original patient cohort at a mean of 10.4 years (7.0 to 15.1) post-operatively. A third of patients with relocated joints had undergone secondary internal rotational osteotomy of the humerus. A mixed effects models approach was used to evaluate the effects of surgery on shoulder rotation, abduction, and the Mallet score. Independent factors were time (pre-and post-surgery), gender, age, joint category (congruent, relocated, relocated plus osteotomy) and whether or not a transfer had been performed. Data from a previously published short-term evaluation were reworked in order to obtain pre-operative values. The mean improvement in external rotation from pre-surgery to the long-term follow-up was 66.5° (95% confidence interval (CI) 61.5 to 71.6). The internal rotation had decreased by a mean of 22.6° (95% CI -18.7 to -26.5). The mean improvement in the three-grade aggregate Mallet score was 3.1 (95% CI 2.7 to 3.4), from 8.7 (95% CI 8.4 to 9.0) to 11.8 (11.5 to 12.1). Our results show that open subscapularis elongation achieves good long-term results for patients with BPBP and an internal rotation contracture, providing lasting joint congruency and resolution of the trumpet sign, but with a moderate mean loss of internal rotation.

Surgical correction of a rotational deformity of the shoulder in patients with obstetric brachial plexus palsy: Short-term results in 270 patients.

We evaluated results at one year after surgical correction of internal rotation deformities in the shoulders of 270 patients with obstetric brachial plexus palsy. The mean age at surgery was 6.2 years (0.6 to 35). Two techniques were used: open subscapularis elongation and latissimus dorsi to infraspinatus transfer. In addition, open relocation was performed or attempted in all patients with subluxed or dislocated joints. A mixed effects model approach was used to evaluate the effects of surgery on internal and external rotation, abduction, flexion and Mallet score. Independent factors included operative status (pre- or post-operative), gender, age, the condition of the joint, and whether or not transfer was performed. The overall mean improvement in external rotation following surgery was 84.6° (95% confidence interval (CI) 80.2 to 89.1) and the mean Mallet score improved by 4.0 (95% CI 3.7 to 4.2). There was a mean decrease in internal rotation of between 27.6° and 34.4° in the relocated joint groups and 8.6° (95% CI 5.2 to 12.0) in the normal joint group. Abduction and flexion were unchanged following surgery. Adding a latissimus dorsi transfer did not result in greater improvement in the mean external rotation compared with elongation of the subscapularis alone.

Properties of motoneurons underlying their regenerative capacity after axon lesions in the ventral funiculus or at the surface of the spinal cord.

Spinal motoneurons represent neurons with axons located in both the central (CNS) and peripheral (PNS) nervous systems. Following a lesion to their axons in the PNS, motoneurons are able to regenerate. The regenerative capacity of these neurons is seen also after lesion in the ventral funiculus of the spinal cord, i.e. within the CNS compartment. Thus, after an axotomy within the ventral funiculus, motoneurons respond with a changing polarity towards production of axons, sometimes even from the dendritic tree. This capacity can be used in cases of ventral root avulsion (VRA) lesions, if a conduit for outgrowing axons is presented in the form of replanted ventral roots. In human cases, this procedure may accomplish return of function in denervated muscles. The strong regenerative capacity of motoneurons provides the basis for studies of the response in motoneurons with regard to their contents of substances related to survival and regeneration. Such studies have shown that, of the large number of receptors for neurotrophic substances and extracellular matrix molecules, mRNAs for receptors or receptor components for neurotrophin-3 (NT-3), ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) are strongly downregulated after VRA, while receptors for glial cell line-derived neurotrophic factor (GDNF) and laminins are profoundly upregulated. These results should be considered in the design of combined pharmacological and surgical approaches to lesions of motor axons at or close to the CNS-PNS interface.

Regulation of laminin-associated integrin subunit mRNAs in rat spinal motoneurons during postnatal development and after axonal injury.

Two important prerequisites for successful axon regeneration are that appropriate extracellular molecules are available for outgrowing axons and that receptors for such molecules are found in the regenerating neuron. Laminins and their receptors in the integrin family are examples of such molecules, and laminin-associated integrin subunits alpha 3, alpha 6, alpha 7, and beta 1 mRNAs have all been detected in adult rat motoneurons. We have here, by use of in situ hybridization histochemistry, examined the normal postnatal development of the expression in motoneurons of these mRNAs and integrin beta 4 mRNA, all of which have been associated with laminin-2. We studied the regulation of these mRNAs, 1-42 days after two types of axotomy in the adult rat (sciatic nerve transection, SNT; ventral root avulsion, VRA) and 1-10 days after SNT in the neonatal animal. During postnatal development, there was a distinct shift in the integrin composition from a stronger expression of the alpha 6 subunit to a very clear dominance of alpha 7 in the adult. All types of axotomy in the adult rat induced initial (1-7 days) large up-regulations of alpha 6, alpha 7 and beta1 subunit mRNAs (250-500%). Only minor changes for alpha 3 mRNA were seen, and beta 4 mRNA could not be detected at all in motoneurons. After adult SNT, the alpha 7 and beta 1 subunits were up-regulated throughout the studied period, and the alpha 6 subunit mRNA was eventually normalized. After VRA, however, the alpha 7 and beta1 levels peaked earlier than after SNT and were normalized at 42 days, whereas alpha 6 mRNA was up-regulated longer than after SNT. Neonatal SNT had much smaller effects on the expression of the studied subunits. The results suggest that an important part of the response to axotomy of motoneurons is to up-regulate receptors for laminin. The developmental shift in integrin subunit composition and the various responses seen in the lesion models indicate that different isoforms of laminin play a role in the regenerative response.

Differential regulation of trophic factor receptor mRNAs in spinal motoneurons after sciatic nerve transection and ventral root avulsion in the rat.

After sciatic nerve lesion in the adult rat, motoneurons survive and regenerate, whereas the same lesion in the neonatal animal or an avulsion of ventral roots from the spinal cord in adults induces extensive cell death among lesioned motoneurons with limited or no axon regeneration. A number of substances with neurotrophic effects have been shown to increase survival of motoneurons in vivo and in vitro. Here we have used semiquantitative in situ hybridization histochemistry to detect the regulation in motoneurons of mRNAs for receptors to ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) 1-42 days after the described three types of axon injury. After all types of injury, the mRNAs for GDNF receptors (GFRalpha-1 and c-RET) and the LIF receptor LIFR were distinctly (up to 300%) up-regulated in motoneurons. The CNTF receptor CNTFRalpha mRNA displayed only small changes, whereas the mRNA for membrane glycoprotein 130 (gp130), which is a critical receptor component for LIF and CNTF transduction, was profoundly down-regulated in motoneurons after ventral root avulsion. The BDNF full-length receptor trkB mRNA was up-regulated acutely after adult sciatic nerve lesion, whereas after ventral root avulsion trkB was down-regulated. The NT-3 receptor trkC mRNA was strongly down-regulated after ventral root avulsion. The results demonstrate that removal of peripheral nerve tissue from proximally lesioned motor axons induces profound down-regulations of mRNAs for critical components of receptors for CNTF, LIF, and NT-3 in affected motoneurons, but GDNF receptor mRNAs are up-regulated in the same situation. These results should be considered in relation to the extensive cell death among motoneurons after ventral root avulsion and should also be important for the design of therapeutical approaches in cases of motoneuron death.

Induction of VEGF and VEGF receptors in the spinal cord after mechanical spinal injury and prostaglandin administration.

Vascular endothelial growth factor (VEGF) is an angiogenetic factor that promotes endothelial cell proliferation during development and after injury to various types of tissue, including the central nervous system (CNS). Using immunohistochemical and in situ hybridization methods we have here demonstrated that VEGF and its receptors Flk-1, Flt-1 and Neuropilin-1 mRNAs and proteins are induced after incisions in the rat spinal cord. The inducible enzyme for prostaglandin synthesis cyclooxygenase-2 (COX-2) is known to be upregulated after spinal injury, cerebral ischemia and to stimulate angiogenesis. To test the hypothesis that prostaglandins may be involved in the VEGF response after lesion we investigated whether intraspinal microinjections of prostaglandin F2alpha (PGF2alpha) alters VEGF expression in the spinal cord. Such treatment was followed by a strong upregulation of VEGF mRNA and protein in the injection area. Finally, by use of an in vitro model with cell cultures of meningeal fibroblast and astrocyte origin, resembling the lesion area cellular content after spinal cord injury but devoid of inflammatory cells, we showed that VEGF is expressed in this in vitro model cell system after treatment with PGF2alpha and prostaglandin E2 (PGE2). These data suggest that cells within a lesion area in the spinal cord are capable of expressing VEGF and its receptors in response to mechanical injury and that prostaglandins may induce VEGF expression in such cells, even in the absence of inflammatory cells.

Neuroprotection by encephalomyelitis: rescue of mechanically injured neurons and neurotrophin production by CNS-infiltrating T and natural killer cells.

In experimental autoimmune encephalomyelitis (EAE), CD4(+) self-reactive T cells target myelin components of the CNS. However, the consequences of an autoaggressive T cell response against myelin for neurons are currently unknown. We herein demonstrate that EAE induced by active immunization with an encephalitogenic myelin basic protein peptide dramatically reduces the loss of spinal motoneurons after ventral root avulsion in rats. Both brain-derived neurotophic factor (BDNF)- and neurotrophin-3 (NT-3)-like immunoreactivities were detected in mainly T and natural killer (NK) cells in the spinal cord. In addition, very high levels of BDNF, NT-3, and glial cell line-derived neurotrophic factor mRNAs were present in T and NK cell populations infiltrating the CNS. Interestingly, bystander recruited NK and T cells displayed similar or higher neurotrophic factor levels compared with the EAE disease-driving encephalitogenic T cell population. High levels of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) mRNAs were also detected, and both these cytokines can be harmful to several types of CNS cells, including neurons. However, treatment of embryonic motoneuron cultures with TNF-alpha or IFN-gamma only had a deleterious effect in cultures deprived of neurotrophic factors. These results suggest that the potentially neurodamaging consequences of severe CNS inflammation are curbed by the production of several potent neurotrophic factors in leukocytes.

Expression of MHC class I heavy chain and beta2-microglobulin in rat brainstem motoneurons and nigral dopaminergic neurons.

We demonstrate here that motoneurons and nigral dopaminergic neurons in the brainstem of the adult rat, with the exception of motoneurons innervating ocular muscles, display high levels of both MHC class I heavy chain and beta2-microglobulin mRNAs. These neurons also display interferon-gamma receptor mRNA. We find it striking that these particular neurons are those which are vulnerable to neurodegeneration in diseases such as Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS).

Expression of insulin-like growth factors and corresponding binding proteins (IGFBP 1-6) in rat spinal cord and peripheral nerve after axonal injuries.

Insulin-like growth factors (IGFs) exert trophic effects on several different cell types in the nervous system, including spinal motoneurons. After peripheral nerve injury, the increased expression of IGFs in the damaged nerve has been suggested to facilitate axonal regeneration. Here we have examined the expression pattern of mRNAs encoding IGF-1 and and -2, IGF binding proteins (IGFBPs) 1-6 in the rat spinal cord and peripheral nerve in three lesion models affecting lumbar motoneurons, i.e., sciatic nerve transection, ventral root avulsion, and a cut lesion in the ventral funiculus of the spinal cord. The expression was also studied in enriched Schwann cell and astrocyte cultures. The injured sciatic nerve expressed IGF-1 and IGF-2 as well as IGFBP-4 and IGFBP-5, whereas central nervous system (CNS) scar tissue expressed IGF-1, IGFBP-2, and IGFBP-5. IGFBP-6 mRNA was strongly upregulated in spinal motoneurons after all three types of lesions. IGFBP-6-like immunoreactivity was present in motoneuron cell bodies, dendrites in the ventral horn, and axons in the sciatic nerve. In line with the in vivo findings, cultured Schwann cells expressed IGF-1, IGF-2, IGFBP-4, and IGFBP-5 mRNAs, whereas cultured astrocytes expressed IGF-1, IGFBP-2, and IGFBP-5 mRNAs. These findings show that IGF-1 is available for lesioned motoneurons both after peripheral and central axonal lesions, whereas there are clear differences in the expression patterns for IGF-2 and some of the binding proteins in CNS and peripheral nervous system (PNS) scar tissue. The robust upregulation of IGFBP-6 mRNA in lesioned motoneurons suggests that this binding protein may be of special relevance for the severed cells.

Expression of MHC class I and beta2-microglobulin in rat spinal motoneurons: regulatory influences by IFN-gamma and axotomy.

The low expression of MHC antigens is believed to be one factor of importance contributing to the immune-privileged status of CNS neurons. We here describe that motoneurons, in contrast to other nerve cells in the lumbar spinal cord of the adult rat, express both MHC class I and beta2-microglobulin mRNA. The motoneurons also display in situ hybridization signal for IFN-gamma receptor mRNA. After a peripheral axotomy, the motoneurons show a clear upregulation of beta2-microglobulin mRNA. IFN-gamma treatment of cultured rat embryonic spinal motoneurons causes a similar upregulation of especially beta2-microglobulin. Based on these facts, we propose that spinal motoneurons can be influenced by IFN-gamma and recognized by cytotoxic CD8+ T-cells. These findings could be of relevance in the search for pathogenetic mechanisms in motoneuron-specific diseases, such as ALS.

Changes in the mRNA expression pattern, with special reference to calcitonin gene-related peptide, after axonal injuries in rat motoneurons depends on age and type of injury.

The axotomy reaction in motoneurons after a peripheral nerve transection in the adult animal is characterized by a robust upregulation of alpha-calcitonin gene-related peptide (CGRP) messenger RNA (mRNA) together with mRNAs encoding cytoskeletal and growth-related proteins. Here we have examined whether the nature of the lesion and the age of the animal have any impact on the mRNA regulation in severed cells. Thus, the effect of a sciatic nerve transection in the adult rat was compared with, on the one hand, ventral root avulsions in the adult animal and, on the other hand, sciatic nerve transection in the immature animal. In the two latter cases, a proportion of the lesioned cells die and overall chances of regeneration are small. In the adult animal a sciatic nerve transection induced an upregulation of alpha-CGRP mRNA from the 3rd day after surgery and throughout the first 3 weeks (the time span of the study). Also low-affinity nerve growth factor receptor (p75) and growth-associated protein-43 (GAP-43) mRNAs were upregulated during the entire 3-week period. In contrast, after ventral root avulsion, the expression of alpha-CGRP, c-jun, and p75 mRNAs were normalized within the 1st postoperative week, while GAP-43 mRNA was still upregulated at 3 weeks. Galanin message-associated peptide (GMAP) mRNA became upregulated preferentially in motoneurons subjected to ventral root avulsion, while nitric oxide synthase (NOS) mRNA was expressed exclusively after the latter type of injury. In the immature animal, alpha-CGRP mRNA was downregulated after sciatic nerve transection in rats aged 3 days or 7 days at the time of surgery; while, in contrast, an upregulation was seen in 12- or 21-day-old animals. GAP-43 and c-jun mRNAs were upregulated in lesioned motoneurons of all ages, while GMAP mRNA was upregulated preferentially in lesioned motoneurons of early postnatal animals. p75 mRNA was expressed in unlesioned immature motoneurons until the age of 7-10 days. The downregulation of p75 mRNA in intact cells at this age coincided with a developmental switch in the ability of axotomized cells to express increased levels of p75 mRNA. No expression of NOS mRNA was detectable in lesioned cells of any of the age groups. These results show that the age of the animal and the type of axonal injury are indeed to a high degree influencing the changes seen in the protein expression pattern in axotomized rat motoneurons. The different responses in these paradigms suggest differences in the trophic response from surrounding glia or the trophic responsiveness of lesioned motoneurons. Also, the results may indicate different roles for the studied substances during the regenerative response of lesioned neurons. Of the substances studied here, upregulation of alpha-CGRP and p75 mRNAs best correlated with a possibility of axon regeneration.

Regulatory effects of trophic factors on expression and distribution of CGRP and GAP-43 in rat motoneurons.

Trophic factors play important roles for the regulation of several motoneuron characteristics. In this report, we have studied the effects of different trophic factors on the regulation of two substances that have been linked with axon sprouting and regeneration in rat spinal motoneurons, i.e., calcitonin gene-related peptide (CGRP) and growth-associated protein-43 (GAP-43). The expression of both GAP-43 and CGRP is regulated developmentally and after different lesions. In the adult animal a sciatic nerve transection normally leads to an increased expression of both alpha-CGRP and GAP-43. Local administration of insulin-like growth factor-1 (IGF-1), IGF-2, or ciliary neurotrophic factor (CNTF) did not significantly change this response, whereas basic fibroblast growth factor (bFGF) attenuated the lesion-induced upregulation of alpha-CGRP mRNA. None of the studied factors had any influence on the upregulation of GAP-43 after lesion. In vitro, GAP-43 and alpha-CGRP mRNAs could be detected in enriched motoneuron cultures prepared from E14 rat embryos. The GAP-43 expression was upregulated by bFGF, IGF-1, IGF-2, and CNTF. CNTF also upregulated alpha-CGRP mRNA in cultured motoneurons, whereas bFGF had the opposite effect. IGF-1 and -2 did not significantly affect the alpha-CGRP mRNA levels. The decrease in GAP-43-immunopositive neuromuscular junctions (NMJs), seen during normal postnatal development, was attenuated after intramuscular injections of bFGF, CNTF, IGF-2, or CGRP 8-37, a CGRP antagonist. At the same time bFGF decreased and CNTF increased the number of CGRP-immunoreactive NMJs, whereas no difference from control was seen in IGF-2-treated muscles. These results show important regulatory effects of trophic factors on the expression of both GAP-43 and alpha-CGRP in motoneurons. However, it also is shown that the trophic factors used here influence the expression of these two substances in distinctively different patterns. This may have important implications for the understanding of trophic interactions in the spinal motor system and also should be considered in the evaluation of possible effects of CGRP and GAP-43 in motoneurons. The effect of a CGRP antagonist on GAP-43 in muscle indicates a role for CGRP in sprouting-related processes.

GDNF mRNA in Schwann cells and DRG satellite cells after chronic sciatic nerve injury.

Glial cell line-derived neurotrophic factor (GDNF) exhibits neurotrophic properties on different types of neurones, including fetal motoneurones and embryonic neurones of sensory ganglia. We demonstrate that chronic injury to the adult rat sciatic nerve induces a rapid up-regulation of GDNF mRNA expression in Schwann cells proximal as well as distal to the injury site, and that expression of this mRNA remains at high levels for at least 5 months after injury. In addition, GDNF mRNA increases and remains high in satellite cells and Schwann cells of the affected L4/L5 DRGs. These findings suggest that GDNF is an important factor in the events that follow upon adult chronic primary sensory neurone injury, and possibly also after adult motoneurone axotomy.

Spinal axons in central nervous system scar tissue are closely related to laminin-immunoreactive astrocytes.

Although transected central nervous system axons fail to regrow after injuries in adult mammals, they send sprouts into the scar tissue that forms at the lesion. We have investigated the relation between scar cells, laminin-like immunoreactivity and cut spinal axons in two previously characterized spinal cord lesion types. Labeling with antisera to glial fibrillary acidic protein and laminin demonstrated that the scar tissue formed after lesions in the rat and cat dorsal and ventral funiculi showed prominent gliosis and strong laminin-like immunoreactivity four days to one year postlesion. Axonal sprouts in the scar, visualized with antibodies to neurofilament (RT97) or by tracing using fluorescein-conjugated dextran, were ensheathed by a thin layer of strongly laminin-immunoreactive tissue. Immunoelectron microscopy demonstrated that axons in the scar were ensheathed predominantly by astrocytes, and that the surface of the cells outlining the axons in the scar showed strong laminin-like immunoreactivity. Adhesive and neurite orienting properties in the scar tissue were assessed in an in vitro system where PC12 cells were cultured on spinal cord slices from dorsal funiculus-lesioned rats. Very few cells adhered to the spinal cord section except for the part where the scar tissue had formed, where numerous cells were attached. The PC12 cells that had adhered to the scar tissue were mainly seen in parts of the scar that showed laminin-like immunoreactivity and their neurites predominantly followed tissue showing laminin-like immunoreactivity. The close association between axonal sprouts and laminin-like immunoreactivity indicates a role for laminin in axonal growth and/or guidance in the injured spinal cord.