Diverse expression of selected SMN complex proteins in humans with sporadic amyotrophic lateral sclerosis and in a transgenic rat model of familial form of the disease.

BACKGROUND AND OBJECTIVE:There is circumstantial evidence linking sporadic amyotrophic lateral sclerosis (ALS) cases to a malfunction or deficit of a multimeric SMN complex that scrutinizes cellular RNAs; the core of this complex is survival motor neuron (SMN, or gemin 1) protein. We intended to verify this hypothesis by comparing the expression of both SMN and several other functionally associated gemins in the anterior horn motoneurons of patients who died of sporadic ALS (sALS), of transgenic rats with overexpression of the mutated human superoxide dismutase 1 gene (SOD1(G93A)) that represent a model of familial ALS (fALS), and of the respective controls. METHODS:Using archival material of paraffin blocks with samples of human and rat spinal cords, immunohistochemical reactions with antibodies against SMN and gemins 2, 3, and 4 were performed and assessed by light microscopy. RESULTS:The expression of SMN and all other studied gemins was observed in motoneurons of sALS patients, fALS rats, and in all controls, although the intensity varied. The immunolabeling was most intense in sALS patients with relatively fast disease course, and decreased with increasing disease duration in both the human sALS and rat fALS material. Irrespective of the disease stage, sALS material showed no or very low gemin 2 immunoreactivity, while clear gemin 2 immunoreactivity was observed in all fALS rats and control material. CONCLUSION:The deficient expression of gemin 2 in spinal cord motoneurons in human sALS may lead to a dysfunction and loss of neuroprotective action of the SMN complex

Nanofiber mat spinal cord dressing-released glutamate impairs blood-spinal cord barrier

An excessive glutamate level can result in excitotoxic damage and death of central nervous system (CNS) cells, and is involved in the pathogenesis of many CNS diseases. It may also be related to a failure of the blood-spinal cord barrier (BSCB). This study was aimed at examining the effects of extended administration of monosodium glutamate on the BSCB and spinal cord cells in adult male Wistar rats. The glutamate was delivered by subarachnoidal application of glutamate-carrying electrospun nanofiber mat dressing at the lumbar enlargement level. Half of the rats with the glutamate-loaded mat application were treated systemically with the histone deacetylase inhibitor valproic acid. A group of intact rats and a rat group with subarachnoidal application of an ‘empty’ (i.e., carrying no glutamate) nanofiber mat dressing served as controls. All the rats were euthanized three weeks later and lumbar fragments of their spinal cords were harvested for histological, immunohistochemical and ultrastructural studies. The samples from controls revealed normal parenchyma and BSCB morphology, whereas those from rats with the glutamate-loaded nanofiber mat dressing showed many intraparenchymal microhemorrhages of variable sizes. The capillaries in the vicinity of the glutamate-carrying dressing (in the meninges and white matter alike) were edematous and leaky, and their endothelial cells showed degenerative changes: extensive swelling, enhanced vacuo­lization and the presence of vascular intraluminal projections. However, endothelial tight junctions were generally well preserved. Some endothelial cells were dying by necrosis or apoptosis. The adjacent parenchyma showed astrogliosis with astrocytic hypertrophy and swelling of perivascular astrocytic feet. Neurons in the parenchyma revealed multiple symptoms of degeneration, including, inter alia, perikaryal, dendritic and axonal swelling, and destruction of organelles. All the damage symptoms were slightly less severe in the rats given valproic acid treatment, and were absent from both the intact rats and the rats with ‘empty’ nanofiber mat dressing. These results demonstrate that glutamate-loaded nanofiber mat dressing can locally create glutamate levels capable of damaging BSCB and that the resulting damage can be mitigated with concurrent systemic valproate treatment

Effects of liposomes with polyisoprenoids, potential drug carriers,on the cardiovascular and excretory system in rats

Background: The unpredictable side effects of a majority currently used drugs are the substantial issue, in which patients and physicians are forced to deal with. Augmenting the therapeutic efficacy of drugsmay prove more fruitful than searching for the new ones. Since recent studies show that new cationic derivatives of polyisoprenoid alcohols (APrens) might exhibit augmenting properties, we intend to use them as a component of liposomal drug carriers. In this study we investigate if these compounds do not per se cause untoward effects on the living organism. Methods: Male Sprague–Dawley rats received for four weeks daily injections (0.5 ml sc) of liposomes built of dioleoyl phosphatidylethanolamine (DOPE), liposomes built of DOPE and APren-7 (ratio 10:1) or water solvent. Weekly, rats were observed in metabolic cages (24 h); blood and urine were sampled for analysis; body weight (BW) and systolic blood pressure (SBP) were determined. After chronic experiment, kidneys and heart were harvested for histological and morphometric analysis. Results: The 4-week BW increments were in the range of 97 ± 4 to 102 ± 4%, intergroup differences were not significant. Microalbuminuria was the lowest in the group receiving liposomes with APren-7 (0.22 ± 0.03 mg/day). Water and food intake, plasma and urine parameters were similar in all groups. Conclusions: Newly designed liposomes containing APren-7 did not affect functions of the excretory and cardiovascular systems, and renal morphology; therefore we find them suitable as a component of liposomal drug carriers

Surface plot profiles of SMN and gemins 2–4 staining in the rat fALS model employed.

<p>Pseudocolors show staining intensity.</p

Changes in number of surviving motoneurons (MNs) in spinal cord ventral horns of transgenic <i>SOD1^G93A</i> male rats over the course of the model disease.

a<p>In relation to the mean MNs number in the transgenic rats 60 days of age;</p><p>**p<0.01,</p><p>***p<0.001;</p><p>one-way ANOVA (F_3,8 = 42.6, p<0.001) followed by Dunnett’s test.</p

Immunostaining for SMN and gemins in the spinal cords of symptomatic fALS rats.

<p>Immunofluorescence shows co-expression of SMN and individual gemins 2–4 in spinal cord motoneurons of >120-day-old fALS rats.</p

SMN and other gemins immunostaining intensity in spinal cord anterior horns motoneurons in sALS patients in relation to the duration of the disease.

a<p>The intensity of specific brown immunostaining was graded as follows:</p><p>+++ - very intense,</p><p>++ - intense,</p><p>+ - moderate,</p><p>+/- - low,</p><p>–/+ - vestigial,</p><p>– - absent.</p

Expression of SMN and gemins 2–4 in the rat fALS model employed.

<p>Note decreasing immunostaining for gemins with the disease duration and progress.</p

Immunostaining of SMN and gemins in the spinal cord of sALS patient.

<p>Immunofluorescence shows co-expression of SMN and individual gemins in spinal cord motoneurons of a 52 years-old patient who died with a 1.5 years history of sALS.</p

Basic clinical features of the ALS patients and controls covered by the study, and numbers of surviving motoneurons (MNs) in the anterior horns of their spinal cord cervical enlargement (C4–C8 level).

a<p>This period included the 2-hour period immediately following the death, which the bodies, as required by the Polish law, spent at room temperature before being transferred to hospital mortuary refrigerator.</p>b<p>In relation to the averaged mean number of surviving MNs in controls.</p