8 research outputs found
Different expression of ECM in uPAR−/− nerves after damage.
<p>Sciatic nerve cryosections from Wt (A, C, E, G, I, K) and uPAR−/− (B, D, F, H, J, L) mice stained for fibrin (Fg), fibronectin (Fn) or vitronectin (Vn), and neurofilaments (Nf) at 15 and 45 dpc. Both at 15 and 45 dpc Fibrin expressions was higher in uPAR−/− as compared to Wt endoneurium (B versus A, and H versus G). Fibronectin expression was similarly in Wt and uPAR−/− mice at both time points (D versus C, and J versus I). Vitronectin expression was higher in uPAR−/− mice as compared to wt at 15dpc (F versus E), whereas it was similar at 45 dpc (L and K). Bar = 50 µm.</p
Fibrinolytic activity in human neuropathies.
<p>In situ zymography in sural nerve biopsies of patients (#4 and #3) with regenerating (A, B) and (#15 and #18) non-regenerating (C, D) neuropathies. Fluorescent signal as a readout of fibrinolytic activity is very high in regenerating nerves, and very low in non-regenerating nerves. A<sup>I</sup> and C<sup>I</sup> are the same reaction depicted in A and C in which fibrinolytic reaction was blocked by amiloride (PAstop). Zym = fibrinolytic activity; Nf = neurofilaments. Bar = 50 µm.</p
Sciatic nerve regeneration after injury.
<p>(A–B, D–E) semithin section and (C, F) fiber type distribution from sciatic nerve of Wt and uPAR null mice at 15 and 45 dpc. At both 15 and 45 dpc we observed reduced number of regenerating fibers. (G) g-ratio was significantly increased in uPAR−/− regenerating fibers at 45 dpc (n. 20000; p = 0.01). (H) Neurophysiological analysis showing similar values of cMAP between Wt and uPAR−/− mice, whereas NCV was significantly reduced in uPAR−/− mice (n. 8; p = 0.001). (I–J) Staining for Mac-1/CD11b (Mac1) in Wt (I) and uPAR null (J) sciatic nerve 45dpc. (K) Quantification of number of macrophages observed in sciatic nerve 15, 21 and 45dpc; differences were significant at 21 and 45 dpc (*p = 0.04; **p = 0.008). Bar = 10 µm in A, B, D, E, I and J.</p
Impaired uPA activity and fibrin clearance in uPAR−/− nerves after damage.
<p>(A) western blot analysis of fibrin and fibronectin in Wt and uPAR null sciatic nerve homogenate at 15, 21 and 45 dpc. Calnexin was used to normalize loading. Quantification of western blot is reported as an average of 3 independent experiments, and represented as ratio fibrin/calnexin, vitronectin/calnexin and fibronectin/calnexin, assigning Wt 0dpc as 1±SEM). At each time point uPAR null homogenate showed increased levels of fibrin and vitronectin as compared to Wt, whereas fibronectin levels were higher at 15 and 21 dpc, but lower at 45 dpc. (B) Zymography of sciatic nerve homogenate from Wt and uPAR null mice measuring tPA and uPA activity 0, 7 and 15dpc. Bands were stained with Coomassie blue as loading control. Quantification of zymography is reported as an average of 3 independent experiments, and represented as ratio uPA/coomassie blue and tPA/coomassie blue, assigning Wt 0dpc as 1±SEM. Note the reduced increase of uPA in uPAR null homogenate as compared to Wt 7 and 15dpc, whereas there are no differences in the tPA activity between mutant and Wt mice. Fg = fibrin; Fn = fibronectin; Vn = vitronectin; Cln = calnexin.</p
Characterization of human nerve biopsies and evaluation of fibrinolytic activity.
<p><b>Legend</b>: CIDP: chronic inflammatory demyelinating neuropathy; CMT: Charcot-Marie-Tooth neuropathy; UCTD: undifferentiated connective tissue disease; ax: axonal; np: neuropathy. FG: fibrin; VN: vitronectin; FN: fibronectin; m: months; y: years.</p><p>*patients already described in <sup>5</sup>, respectively pt. #3, 6, 7, 8, 11, 12, 14, 17, 19, 20, 39, 41, 27, 30, 43.</p
Fibrinolytic molecules in myelination.
<p>(A) Time course zymography of uPA and tPA activity in Wt DRG explant homogenate (as a pool of at least 8 coverslips) and their conditioned media without ascorbic acid treatment (-) or after 5, 10, 15, 20 days of ascorbic acid (+). Note both tPA and uPA activity are induced after ascorbic acid in both cell homogenate and media, although uPA activity increases in parallel with myelination. (B–E) DRG explants from Wt and uPAR null mice stained for neurofilament (green) and MBP (red) 7 days after ascorbic acid. The number of myelinated segments were similar between Wt and uPAR null explants. Bar = 50 µm.</p
Histological characteristics of uPAR null nerve.
<p>(A–B) semithin sections from sciatic nerve of Wt (A) and uPAR null mouse (B) showing normal fiber appearance. (C) Similar fiber type distribution in Wt and uPAR null sciatic nerve. (D) Neurophysiology analysis showing similar values of cMAP and NCV between Wt and uPAR−/− mice (n. 10 mice per group). (E) g-ratio did not show differences in myelin thickness between Wt and uPAR−/− nerves (n. 20000). (F–K) sciatic nerve cryosections of Wt and uPAR null mouse stained for fibrin fibronectin and vitronectin. Fibrin and vitronectin staining was mildly increased in uPAR null endoneurium (G and K) as compared to Wt (F and J), whereas fibronectin was similarly expressed (H–I). (L) Western blot analysis of fibrin and fibronectin in Wt and uPAR null sciatic nerve homogenate. Calnexin was used to normalize loading (fibronectin and vitronectin were loaded on the same gel, hence they have the same calnexin bands). r = densitometric ratio between the band of interest and calnexin; Wt was always assigned as r = 1. Fibrin and vitronectin levels were increased in uPAR null nerves as compared to Wt, whereas levels of fibronectin were similar. Fg = fibrin; Fn = fibronectin; Vn = vitronectin; Nf = neurofilaments. Bar = 15 µm in A and B; 50 µm in F–K.</p
DataSheet_1_Clinical and pathological findings in neurolymphomatosis: Preliminary association with gene expression profiles in sural nerves.docx
Although inflammation appears to play a role in neurolymphomatosis (NL), the mechanisms leading to degeneration in the peripheral nervous system are poorly understood. The purpose of this exploratory study was to identify molecular pathways underlying NL pathogenesis, combining clinical and neuropathological investigation with gene expression (GE) studies. We characterized the clinical and pathological features of eight patients with NL. We further analysed GE changes in sural nerve biopsies obtained from a subgroup of NL patients (n=3) and thirteen patients with inflammatory neuropathies as neuropathic controls. Based on the neuropathic symptoms and signs, NL patients were classified into three forms of neuropathy: chronic symmetrical sensorimotor polyneuropathy (SMPN, n=3), multiple mononeuropathy (MN, n=4) and acute motor-sensory axonal neuropathy (AMSAN, n=1). Predominantly diffuse malignant cells infiltration of epineurium was present in chronic SMPN, whereas endoneurial perivascular cells invasion was observed in MN. In contrast, diffuse endoneurium malignant cells localization occurred in AMSAN. We identified alterations in the expression of 1266 genes, with 115 up-regulated and 1151 down-regulated genes, which were mainly associated with ribosomal proteins (RP) and olfactory receptors (OR) signaling pathways, respectively. Among the top up-regulated genes were actin alpha 1 skeletal muscle (ACTA1) and desmin (DES). Similarly, in NL nerves ACTA1, DES and several RPs were highly expressed, associated with endothelial cells and pericytes abnormalities. Peripheral nerve involvement may be due to conversion towards a more aggressive phenotype, potentially explaining the poor prognosis. The candidate genes reported in this study may be a source of clinical biomarkers for NL.</p