Localization of N-type Ca2+ channels in the rat spinal cord following chronic constrictive nerve injury

Previous studies have shown that spinal L-type, N-type, and P-type Ca²⁺-channel blockers are effective in modulating pain behavior caused nerve injury. In the present work, using the loose ligation of the sciatic nerve model, we characterized the time course of the appearance of tactile and cold allodynia and the corresponding spinal expression of the N-type Ca²⁺ channel α_1B-subunit after nerve ligation. Within 1 week after ligation, the majority of rats developed a unilateral sensitivity to mechanical stimulation (von Frey filaments), as well as sensitivity to cold, which persisted for 30 days. Immunocytochemical analysis of the spinal cord in sham-operated animals for the α_1B-subunit showed a smooth, moderate staining pattern in the superficial laminae I–II, as well as in ventral α-motoneurons. In nerve-ligated animals, an intense, dot-like immunoreactivity in the ipsilateral dorsal horn was observed from 5–20 days after nerve ligation. The most prominent α_1B-subunit upregulation was found in the outer as well as the inner part of lamina II (II_o, II_i), extending from the medial toward the lateral region of the L4 and L5 spinal segments. The behavioral changes which developed after chronic constriction injury directly correlated with the α_1B-subunit upregulation in the corresponding spinal cord segments. These data suggest that upregulation of the spinal α_1B-subunit may play an important role in the initiation and maintenance of pain state after peripheral nerve injury. Electronic Publication     

Gaucher's disease with Parkinson's disease: clinical and pathological aspects

The association between type 1 Gaucher disease and PD has been reported in the literature. The clinical picture is characterized by the predominance of bilateral akinetic-rigid signs and poor response to levodopa therapy. The authors describe four patients (two siblings) with type 1 Gaucher disease presenting with the following signs of typical PD: asymmetric onset of rigidity, resting tremor, bradykinesia, and a favorable response to Parkinson therapies.     

Peripheral axotomy affects nicotinamide adenine dinucleotide phosphate diaphorase and nitric oxide synthases in the spinal cord of the rabbit

Using nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry and nitric oxide synthase (NOS) immunocytochemistry combined with radioassay of calcium-dependent NOS activity, we examined the occurrence of NADPHd staining and NOS immunoreactivity (NOS-IR) in the dorsal root ganglia (DRG) neurons, dorsal root afferents, and axons projecting via gracile fascicle to gracile nucleus 14 days after unilateral sciatic nerve transection in the rabbit. Mild to moderate NADPHd staining and NOS-IR appeared in a large number of small and medium-sized to large neurons in the ipsilateral L4-L6 DRG, accompanied by enhanced NOS-IR of thick myelinated fibers in the ipsilateral L4-L6 dorsal roots. A noticeable increase in the density of punctate NADPHd staining occurred throughout laminae I-IV in the ipsilateral medial part of the dorsal horn in L4-L6 segments. Concurrently, a statistically significant decrease in the number of small NADPHd-exhibiting neurons in laminae I-II and, in contrast to this, a statistically significant increase of medium-sized to large NADPHd-stained somata in the ipsilateral laminae III-VI of L4-L6 segments were found. A detailed compartmentalization of L4-L6 segments into gray and white matter regions disclosed substantially increased catalytic NOS activity and inducible NOS mRNA levels in the dorsal horn and dorsal column ipsilaterally to the peripheral injury. A noticeable increase in the number of thick myelinated NADPHd-exhibiting and NOS-IR axons was noted in the ipsilateral gracile fascicle, terminating in dense, punctate NADPHd staining in the neuropil of the gracile nucleus. These observations indicate that the de novo-synthesized NOS in the lesioned primary afferent neurons resulting after sciatic nerve transection may be involved in an increase in NADPHd staining and NOS-IR in the ipsilateral dorsal roots and dorsal horn of L4-L6 segments, whence NOS could be supplied to ascending axons of the gracile fascicle.     

ALS-linked TDP-43 mutations produce aberrant RNA splicing and adult-onset motor neuron disease without aggregation or loss of nuclear TDP-43

Transactivating response region DNA binding protein (TDP-43) is the major protein component of ubiquitinated inclusions found in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with ubiquitinated inclusions. Two ALS-causing mutants (TDP-43^Q331K and TDP-43^M337V), but not wild-type human TDP-43, are shown here to provoke age-dependent, mutant-dependent, progressive motor axon degeneration and motor neuron death when expressed in mice at levels and in a cell type-selective pattern similar to endogenous TDP-43. Mutant TDP-43-dependent degeneration of lower motor neurons occurs without: (i) loss of TDP-43 from the corresponding nuclei, (ii) accumulation of TDP-43 aggregates, and (iii) accumulation of insoluble TDP-43. Computational analysis using splicing-sensitive microarrays demonstrates alterations of endogenous TDP-43–dependent alternative splicing events conferred by both human wild-type and mutant TDP-43^Q331K, but with high levels of mutant TDP-43 preferentially enhancing exon exclusion of some target pre-mRNAs affecting genes involved in neurological transmission and function. Comparison with splicing alterations following TDP-43 depletion demonstrates that TDP-43^Q331K enhances normal TDP-43 splicing function for some RNA targets but loss-of-function for others. Thus, adult-onset motor neuron disease does not require aggregation or loss of nuclear TDP-43, with ALS-linked mutants producing loss and gain of splicing function of selected RNA targets at an early disease stage.Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) are progressive, adult-onset neurodegenerative diseases with overlapping clinical and pathological features (1–3). ALS is characterized by the selective loss of upper and lower motor neurons, leading to progressive fatal paralysis and muscle atrophy. A large majority (∼90%) of ALS and FTLD-U cases are without a known genetic cause. Importantly, in these sporadic cases, the appearance of ubiquitinated inclusions within the affected neurons of the nervous system characterizes both ALS and FTLD-U patients, suggesting an overlapping mechanism underlying both diseases. Biochemical characterization of brains and spinal cords from ALS and FTLD-U patients identified transactivating response region (TAR) DNA binding protein (TDP-43) as the major protein component of these ubiquitinated inclusions (4, 5). The discovery of ALS-linked mutations in the glycine-rich C-terminal domain of TDP-43 (6-8) demonstrated a pathological role of TDP-43 in both diseases. The subsequent identification of mutations in a structurally and functionally related nucleic acid binding protein, FUS/TLS (fused in sarcoma/translocated in liposarcoma) (9, 10), further implicated defects in RNA processing in ALS pathogenesis.TDP-43 is a multifunctional nucleic acid binding protein. Within the nervous system, TDP-43 binds to >6,000 pre-mRNAs and affects the levels of ∼600 mRNAs and the splicing patterns of another 950 (11). Structurally, the 414-aa protein consists of two RNA recognition motifs (RRM1 and RRM2) (12, 13), nuclear import and export signal (14), and a glycine-rich region implicated in protein–protein interactions (15, 16) that include components of the RNA splicing machinery (17, 18).Disruption in mice of the highly conserved Tardbp gene is embryonically lethal (19–22). Similarly, postnatal inactivation of Tardbp (by Cre recombinase-mediated gene excision encoded by a ubiquitously-expressed CAG-Cre transgene) results in rapid postnatal death accompanied by defects in fat metabolism (22). TDP-43 autoregulates its own RNA level (11, 23) at least in part by stimulating excision of an intron in its 3′ untranslated region, thereby making the spliced RNA a substrate for nonsense-mediated RNA degradation (11). Furthermore, transgenic rodent models have been used to demonstrate that overriding the autoregulatory mechanism by overexpression of unregulated wild-type (24–28) or disease-linked mutant (26, 28–35) TDP-43 transgenes can produce neurodegeneration in mice.ALS and FTLD-U patient brain and spinal cord samples are characterized by the accumulation of cytoplasmic TDP-43 aggregates accompanied by a distinct clearing of nuclear TDP-43 within affected neurons and glia (36, 37), implicating possible loss of nuclear TDP-43 function in disease pathogenesis. In human disease, TDP-43 has been reported to be abnormally phosphorylated, ubiquitinated, and cleaved to produce C-terminal fragments (4, 5, 38, 39). Ectopic expression of these C-terminal fragments in cell-culture models (40–42) has shown that they are aggregation-prone and confer an intrinsic toxicity. However, the extent of the contribution of these C-terminal fragments to disease pathogenesis is undetermined. Indeed, double-immunofluorescent labeling of ALS patient spinal cords using N-terminal–specific and C-terminal–specific antibodies suggests that inclusions in spinal cord motor neurons are comprised primarily of full-length TDP-43 (37). Importantly, retention of ability to bind RNA by full-length TDP-43 has been demonstrated to be required for toxicity in yeast, fly, and Caenorhabditis elegans models (43–46).Nevertheless, it remains unresolved whether toxicity to motor neurons from mutations in TDP-43 is mediated through a gain of toxic property, loss-of-function, or a combination of both. By generation of transgenic mice encoding levels of wild-type or mutant human TDP-43 comparable to endogenous TDP-43, we demonstrate mutant-dependent, age-dependent motor neuron disease from ALS-linked TDP-43 mutants in the absence of overexpression, cytoplasmic accumulation of a 35 kDa TDP-43 fragment, or insoluble TDP-43 aggregates. Accompanying autoregulation-mediated reduction of endogenous wild-type TDP-43 are splicing alterations previously identified to be TDP-43–dependent (11). Additional splicing alterations are identified by systematic genome-wide analyses of alternative splicing that are indicative of both enhancement and loss-of-function by the TDP-43 mutants for individual RNA substrates, from which we conclude that ALS-linked mutations confer both loss- and gain-of-function properties to TDP-43, and that these act intranuclearly to induce splicing alterations that may underlie age-dependent motor neuron disease.     

Botulinum toxin type A: an effective treatment to restore phonation in laryngectomized patients unable to voice

We evaluated the efficacy of Botulinum toxin type A (BTXA) as an alternative to surgical intervention to facilitate phonation in 34 laryngectomized patients (31 males and 3 women) who were unable to produce tracheoesophageal voice because of spasm of the middle and inferior pharyngeal constrictor muscles (PCM). EMG was recorded to confirm activity in these muscles during attempted vocalization. Parapharyngeal nerve block (Carbocaine 2%, 5 cc) was used to demonstrate short-term fluent voice after relaxation of the pharyngeal constrictor muscle. At a later occasion, 100 U of Botox (Allergan) in ten patients and 50 U in two patients were injected unilaterally at one location in the PCM percutaneously under EMG guidance. All patients then underwent a voice therapy program. In 11 out of 12 patients an improvement of phonation was evident after 24–48 h and it was long lasting. This result was also seen in a patient previously myotomized without improvement. Only one patient needed to be reinjected every 3 months. At a follow-up after 3 months the EMG recorded in four patients showed a low-amplitude or complete absence of activity in the treated muscle. No side effects developed. BTX therapy, especially when associated with the speech therapy, is efficacious in restoring voice to laryngectomees who are unable to voice because of spasm of the PCMs. Our results confirm previous reports. This method is our approach of choice in managing PCM spasm because it is non-invasive, not painful, has few or no side effects, and is frequently long-lasting.     

Cluster headache patients are not affected by restless legs syndrome: an observational study

Objective

To investigate the presence of Restless Legs Syndrome (RLS) in Cluster Headache (CH) patients compared to headache-free controls.

Design and setting

Cross-sectional case-control study of CH patients presenting at tertiary headache centers over the period January-December 2008.

Patients and participants

Fifty consecutive patients (6 women and 44 men) of mean age of 39.7 year (standard deviation 10.9) with episodic or chronic CH diagnosed according to ICHD-II criteria and 50 headache-free subjects matched by age and sex were recruited.

Results

None of the CH patients had RLS. Six (12%) headache-free controls had RLS.

Conclusions

Our data indicate no probable relationship between CH and RLS. However, since both conditions have a circadian rhythm and are associated with altered melatonin secretion, we conjecture that reduced nocturnal melatonin in CH likely allows sustained dopaminergic activity which could be protective against RLS in CH patients.     

Vaccination against the 2009 pandemic influenza A (H1N1) among healthcare workers in the major teaching hospital of Sicily (Italy)

The aim of the study was to investigate factors involved in vaccination acceptance among healthcare workers (HCWs) and adverse reactions rates associated with pandemic influenza vaccination. The study was carried out in the major teaching hospital of Sicily from November 2009 to February 2010 on 2267 HCWs. A total of 407 (18%) HCWs were vaccinated against the 2009 pandemic influenza A (H1N1). A logistic regression analysis indicates an increased risk of non-vaccination against pandemic influenza in females (OR = 1.6; 95% CI = 1.3-2.1) compared to males, in nurses/technicians/administrative workers (OR = 1.7; 95% CI = 1.3-2.2) compared to doctors/biologists, and in HCWs who were non-vaccinated against seasonal influenza in 2008-2009 (OR = 4.9; 95% CI = 3.7-6.5) compared to vaccinated HCWs. Overall, 302 (74.2%) out of 407 questionnaires distributed to vaccinated HCWs were returned within the observation period. One hundred fifty-two workers (50.3%) experienced at least one adverse reaction (30.1%, local reactions; 6.6% systemic reactions and 13.6% both of them). The most frequent side effect of vaccination was pain at the injection site (43.4%). Twelve (3.9%) out of 302 HCWs stated they experienced influenza-like illness episodes during the follow-up period. The use of an adjuvanted vaccine against pandemic influenza A (H1N1) appears to be an effective and safe preventive strategy, showing a prevalence of both local and systemic adverse reactions not very different from that seen after vaccination with non-adjuvanted seasonal influenza vaccine. Despite this finding, vaccination coverage among HCWs remains very low, suggesting the need to implement educational campaigns directed to groups with lower coverage rates.