A DAP12-dependent signal promotes pro-inflammatory polarization in microglia following nerve injury and exacerbates degeneration of injured neurons.

Under pathological conditions, activated microglia play paradoxical roles and could have neurotoxic or neuroprotective effects. However, the signal determining how activated microglia affects the fate of neuronal cells remains largely unknown. Here we demonstrate that DNAX-activating protein of 12 kDa (DAP12), a transmembrane adaptor protein that contains an immunoreceptor tyrosine-based activation motif, is a critical regulator of microglial function after nerve injury. In a model of mouse hypoglossal nerve injury, the duration of microglial increase after nerve injury became shorter in mice lacking DAP12, although microglial morphology and total cell numbers were not significantly affected during early phase after nerve injury. Intriguingly, expressions of M1-phenotype markers including pro-inflammatory cytokines were suppressed in DAP12-deficient microglia. Furthermore, axotomy-induced motor neuron death was markedly prevented in DAP12-deficient mice. Collectively, DAP12-mediated microglial activation following axotomy promotes pro-inflammatory responses, and thereby accelerates nerve injury-induced neuron death, suggesting that DAP12 is a potential therapeutic target for the protection of neuronal degeneration caused by microglial activation.

[Hypoglossal nerve injury following endotracheal intubation]. / Uitval van de N. hypoglossus na orotracheale intubatie.

BACKGROUND: Endotracheal intubation is required to keep the airway open when a patient undergoes surgery under general anaesthetic. Here we present a rare complication of endotracheal intubation. CASE: A 70-year-old woman underwent repeat pulmonary vein isolation for atrial fibrillation under general anaesthetic. Because the procedure was expected to take a long time, and because the surgeon might want to perform transoesophageal echocardiography, we chose to carry out endotracheal intubation on this patient. After the operation she showed dyspnoea, problems with swallowing and dysarthria. Physical examination showed elevation of the right pharyngeal arch and deviation of the tongue to the right. This was found to be due to hypoglossal nerve injury. CONCLUSION: Hypoglossal nerve injury is a rare complication of endotracheal intubation. There is no proven effective treatment for this complication. Prognosis is favourable: 69% of the patients recover completely in the first 6 months following intubation. Patients with this complication should receive supportive therapy, such as speech therapy and dietary modifications, to prevent aspiration.

Effects of various lengths of hypoglossal nerve resection on motoneuron survival.

We employed stereological analyses for whole quantification of hypoglossal (XII) motoneurons in adult rats that received varying degrees of resection of the XII nerve. Various lengths of nerve gaps (0.0-13.3 mm) were made at the main trunk of the unilateral XII nerve, and the total number of XII neurons on the injured and uninjured sides was counted 12 weeks after nerve resection. The stereologically estimated total number of XII neurons decreased after various lengths of nerve resection, and survival rates ranged from 34.4% to 87.1%. Statistically significant negative correlations were observed between increasing length of the resected nerve and decreasing XII neuron survival. It was concluded that the total number of XII neurons decreased after nerve resection and that survival rates of XII neurons were related to distances between resected nerve stamps.

SIRT1 activation with neuroheal is neuroprotective but SIRT2 inhibition with AK7 is detrimental for disconnected motoneurons.

Sirtuin 1 (SIRT1) activity is neuroprotective, and we have recently demonstrated its role in the retrograde degenerative process in motoneurons (MNs) in the spinal cord of rats after peripheral nerve root avulsion (RA) injury. SIRT2 has been suggested to exert effects opposite those of SIRT1; however, its roles in neurodegeneration and neuron response after nerve injury remain unclear. Here we compared the neuroprotective potentials of SIRT1 activation and SIRT2 inhibition in a mouse model of hypoglossal nerve axotomy. This injury induced a reduction of around half MN population within the hypoglossal nucleus by a non-apoptotic neurodegenerative process triggered by endoplasmic reticulum (ER) stress that resulted in activation of the unfolded protein response mediated by IRE1α and XBP1 by 21 days post injury. Both SIRT1 activation with NeuroHeal and SIRT2 inhibition with AK7 protected NSC-34 motor neuron-like cells against ER stress in vitro. In agreement with the in vitro results, NeuroHeal treatment or SIRT1 overexpression was neuroprotective of axotomized hypoglossal MNs in a transgenic mouse model. In contrast, AK7 treatment or SIRT2 genetic depletion in mice inhibited damaged MN survival. To resolve the in vitro/in vivo discrepancies, we used an organotypic spinal cord culture system that preserves glial cells. In this system, AK7 treatment of ER-stressed organotypic cultures was detrimental for MNs and increased microglial nuclear factor-κB and the consequent transcription of cytotoxic pro-inflammatory factors similarly. The results highlight the importance of glial cells in determining the neuroprotective impact of any treatment.

Stereological assessment of the total number of hypoglossal neurons after repeated crush injuries to the hypoglossal nerve in adult rats.

OBJECTIVE: Retrograde neuronal cell death does not occur in mature motoneurons following the axonal injury of peripheral nerves. However, a previous study suggested that retrograde neuronal cell death does occur in adult rats after the creation of double lesions on the hypoglossal (XII) nerve based on a substantial decrease in the number of XII neurons. Using stereological methods, we examined neuronal apoptosis in XII neurons and the total number of XII neurons following repeated crush injuries to the XII nerve. METHODS: The right XII nerve of adult rats was crushed three times at one-week intervals with a brain aneurysm clip. At 4 weeks after the final crush, the total numbers of XII neurons on the injured right and uninjured left sides were estimated stereologically. RESULTS: After repeated crush injuries, no apoptosis was evident in XII neurons as indicated by immunostaining for cleaved caspase-3. Moreover, immunohistochemistry for the vesicular acetylcholine transporter revealed axonal elongation in the tongue 4 weeks after repeated crush injuries. At 4 weeks, the total numbers of XII neurons were 7800 ± 290 on the injured right side and 8000 ± 230 on the uninjured left side, and no significant difference was evident between the injured and uninjured sides. CONCLUSION: Neuronal cell death does not occur in XII neurons and the total number of XII neurons does not decrease after repeated crush injuries of the XII nerve in adult rats.

microRNA-124 is down regulated in nerve-injured motor neurons and it potentially targets mRNAs for KLF6 and STAT3.

MicroRNA (miRNA) is a small non-coding RNA that regulates gene expression by degrading target mRNAs or inhibiting translation. Although many miRNAs play important roles in various conditions, it is unclear whether miRNAs are involved in motor nerve regeneration. In this study, we identified the possible implication of miR-124 in nerve regeneration using a mouse hypoglossal nerve injury model. The significant down-regulation of miR-124 was observed in injured hypoglossal motor neurons after nerve injury, and this transient down-regulation showed a clear inverse correlation with the up-regulation of KLF6 and STAT3, known as axon elongation factor and regeneration-associated molecules, respectively. Furthermore, the luciferase assay and in vitro gain of function methods supported that both genes could be potent targets of miR-124. These results suggest that injury-induced repression of miR-124 may be implicated in the regulation of expression of several injury-associated transcription factors, which are crucial for appropriate nerve regeneration.

Novel objective classification of reactive microglia following hypoglossal axotomy using hierarchical cluster analysis.

A total of 136 microglia were intracellularly labeled and their morphological features were evaluated by 3D morphometric measurement. According to hierarchical cluster analysis, microglia were objectively categorized into four groups termed types I-IV. The validity of this classification was confirmed by principal component analysis and linear discriminant analysis. Type I microglia were found in sham-operated mice and in mice sacrificed 28 days (D28) after axotomy. The appearance of type I cells was similar to so-called ramified microglia in a resting state. Type II microglia were mainly seen in D14 mice, which exhibited small cell bodies with thin and short processes. Interestingly, none of the already-known morphological types of microglia seemed to be comparable to type II cells. We thus named type II microglia "small ramified" cells. Types III and IV microglia were mainly seen in D3 and D7 mice and their appearances were similar to hypertrophied and bushy cells, respectively. Proliferating cell nuclear antigen (PCNA), a mitosis marker, was almost exclusively expressed in D3 mice. On the other hand, voltage-dependent potassium channels (Kv1.3/1.5), neurotoxicity-related molecules, were most highly expressed in D14 mice. Increased expression of Kv1.3/1.5 in D14 mice was suppressed by minocycline treatment. These findings indicate that type II and III microglia may be involved in neurotoxicity and mitosis, respectively. Type IV microglial cells are assumed to be in the process of losing mitotic activity and gaining neurotoxicity. Our data also suggest that type II microglia can be a potential therapeutic target against neurodegenerative diseases.

Tapia's syndrome after arthroscopic shoulder stabilisation under general anaesthesia and LMA.

BACKGROUND: Anaesthetic complications, albeit rare, still occur and may be severe and unanticipated, with significant morbidity. Extracranial ipsilateral palsy of the recurrent laryngeal and the hypoglossal nerves is known as the Tapia's syndrome. Damage to these nerves may result from displacement of the head during mask ventilation, endotracheal intubation, bronchoscopy or the use of a laryngeal mask airway (LMA). We describe unilateral paralysis of the muscles of the tongue and ipsilateral vocal cord due to a lesion of cranial nerves X and XII that occurred following LMA anaesthesia combined with plexus block. CASE REPORT: A 57-year-old man with a rupture of the right shoulder underwent arthroscopic shoulder stabilisation and internal fixation. General anaesthesia with aLMA was combined with an interscalene plexus block. After induction with propofol and fentanyl, a LMA was inserted with some difficulty without muscle relaxation. The cuff was inflated with 30 mL of air and further volumes of air until a "just-seal" pressure was obtained. The anaesthesia was maintained with sevoflurane in oxygen/air. The procedure was carried out in a semi-supine position with the head inclined slightly forward, and the upper body slightly elevated. Surgery lasted 55 min and anaesthesia 70 min. After surgery, the patient quickly regained consciousness and the LMA was removed when he was responding to commands and was able to fully open his mouth. During the immediate postoperative period, the patient's voice was hoarse but he breathed without difficulty. The following day, he developed dysphagia and slurred speech; on examination, paralysis of the left side of the tongue was found. The diagnosis of an acute injury to the hypoglossal and laryngeal recurrent nerves was made and the patient was transferred to the neurology clinic for further treatment. CONCLUSIONS: This rare complication reminds us not only of the importance of positioning during anaesthesia and surgery, but also of the need for careful and correct airway management. It could be probably prevented by careful insertion of an appropriate size LMA, and the use of low intracuff pressures and/or volumes.

Proliferation, migration and differentiation of ependymal region neural progenitor cells in the brainstem after hypoglossal nerve avulsion.

PURPOSE: Cells in the ependymal region in the adult central nervous system (CNS) have been found to possess neural progenitor cell (NPC) like features including capacity for generating new neurons and glia in response to injury and inflammatory disease. Whether these cells are activated after a peripheral nerve injury has not previously been extensively evaluated. METHODS: We investigate the possible activation and effect of NPCs in the ependymal region in the immediate vicinity to the hypoglossal nucleus in the brainstem using two models of injuries, hypoglossal nerve transection and nerve avulsion after which the proliferation, migration and differentiation of ependymal regional NPCs were evaluated. RESULTS: We showed that: (i) immunoreactivity for Sox2 was detected in cells in the ependymal region of the brainstem and that BrdU/Sox2-positive cells were observed after avulsion, but not after transection injury; (ii) avulsion induces re-expression of nestin in the ependymal layer as well as induced NPC migration from the ependymal layer; (iii) the chemokine SDF-1α (a marker for migrating cells) was upregulated ipsilateral to the nerve injury; (iiii) the NPCs migrating differentiated only into GFAP-positive astrocytes in the hypoglossal nucleus. CONCLUSION: These results suggest that nerve avulsion injury induces in parallel with the retrograde "axon reaction" activation of endogenous NPCs in the ependymal region and further suggest that these cells could be involved in repair and neuroregeneration after injury within the brainstem.

Differential involvement of perineuronal astrocytes and microglia in synaptic stripping after hypoglossal axotomy.

Following peripheral axotomy, the presynaptic terminals are removed from lesioned neurons, that is synaptic stripping. To elucidate involvement of astrocytes and microglia in synaptic stripping, we herein examined the motoneuron perineuronal circumference after hypoglossal nerve transection. As reported previously, axotomy-induced slow cell death occurred in C57BL/6 mice but not in Wistar rats. Synaptophysin labeling in the hypoglossal nucleus exhibited a minor reduction in both species after axotomy. Slice patch recording showed that the mean frequency of miniature postsynaptic currents in axotomized motoneurons was significantly lower in rats than in mice. We then estimated the relative coverage of motoneuron perineuronal circumference by line profile analysis. In the synaptic environment, axotomy-induced intrusion of astrocytic processes was significantly more extensive in rats than in mice, whereas microglial intrusion into the synaptic space was significantly more severe in mice than in rats. Interestingly, in the extrasynaptic environment, the prevalence of contact between astrocytic processes and lesioned motoneurons was significantly increased in rats, while no significant axotomy-induced alterations in astrocytic contact were observed in mice. These findings indicate that astrocytic, but not microglial, reaction may primarily mediate some anti-apoptotic effects through synaptic stripping after hypoglossal nerve axotomy. In addition, enlargement of astrocytic processes in the extrasynaptic environment may also be involved in neuronal protection via the increased uptake of excessive glutamate.

Alterations in neuronal survival and glial reactions after axotomy by ceftriaxone and minocycline in the mouse hypoglossal nucleus.

Some antibiotics are suggested to exert neuroprotective effects via regulation of glial responses. Attenuation of microglial activation by minocycline prevents neuronal death in a variety of experimental models for neurological diseases, such as cerebral ischemia, Parkinson's and Huntington's disease. Ceftriaxone delays loss of neurons in genetic animal models of amyotrophic lateral sclerosis through upregulation of astrocytic glutamate transporter expression (GLT-1). However, it remains largely unknown whether these antibiotics are able to protect neurons in axotomy models for progressive motor neuron diseases. Recent studies have shown that the axotomized motoneurons of the adult rat can survive, whereas those of the adult mouse undergo neuronal degeneration. We thus examined the possible effects of ceftriaxone and minocycline on neuronal loss and glial reactions in the mouse hypoglossal nucleus after axotomy. The survival rate of lesioned motoneurons at 28 days after axotomy (D28) was significantly improved by ceftriaxone and minocycline treatment. There were no significant differences in the cellular densities of astrocytes between ceftriaxone-treated and saline-treated animals. Ceftriaxone administration increased the expression of GLT-1 in the hypoglossal nucleus, while it suppressed the reactive increase of glial fibrillary acidic protein (GFAP) expression to control level. The cellular densities of microglia at D28 were significantly lower in minocycline-treated mice than in saline-treated mice. The time course analysis showed that immediate increase in microglia at D3 and D7 was not suppressed by minocycline. The present observations show that minocycline and ceftriaxone promote survival of lesioned motoneurons in the mouse hypoglossal nucleus, and also suggest that alterations in glial responses might be involved in neuroprotective actions of antibiotics.