Development of less invasive neuromuscular electrical stimulation model for motor therapy in rodents

Background

Combination therapy is essential for functional repairs of the spinal cord. Rehabilitative therapy can be considered as the key for reorganizing the nervous system after spinal cord regeneration therapy. Functional electrical stimulation has been used as a neuroprosthesis in quadriplegia and can be used for providing rehabilitative therapy to tap the capability for central nervous system reorganization after spinal cord regeneration therapy.

Objective

To develop a less invasive muscular electrical stimulation model capable of being combined with spinal cord regeneration therapy especially for motor therapy in the acute stage after spinal cord injury.

Methods

The tibialis anterior and gastrocnemius motor points were identified in intact anesthetized adult female Fischer rats, and stimulation needle electrodes were percutaneously inserted into these points. Threshold currents for visual twitches were obtained upon stimulation using pulses of 75 or 8 kHz for 200 ms. Biphasic pulse widths of 20, 40, 80, 100, 300, and 500 µs per phase were used to determine strength–duration curves. Using these parameters and previously obtained locomotor electromyogram data, stimulations were performed on bilateral joint muscle pairs to produce reciprocal flexion/extension movements of the ankle for 15 minutes while three-dimensional joint kinematics were assessed.

Results

Rhythmic muscular electrical stimulation with needle electrodes was successfully done, but decreased range of motion (ROM) over time. High-frequency and high-amplitude stimulation was also shown to be effective in alleviating decreases in ROM due to muscle fatigue.

Conclusions

This model will be useful for investigating the ability of rhythmic muscular electrical stimulation therapy to promote motor recovery, in addition to the efficacy of combining treatments with spinal cord regeneration therapy after spinal cord injuries.     

Regulation of protein synthesis in rabbit reticulocyte lysates: Additional initiation factor required for formation of ternary complex (eIF-2·GTP·Met-tRNAf) and demonstration of inhibitory effect of heme-regulated protein kinase

Heme deficiency in rabbit reticulocytes and their lysates leads to the activation of a heme-regulated translational inhibitor (HRI) which causes the cessation of polypeptide initiation. HRI is a protein kinase that specifically phosphorylates the 38,000-dalton subunit of eukaryotic initiation factor 2 (eIF-2). eIF-2 binds Met-tRNA(f) and GTP in ternary complex. As a continuation of the studies on the molecular basis of the inhibition of the formation of 40S ribosomal subunit-Met-tRNA(f) complexes by HRI [Ranu, R. S., London, I. M., Das, A., Dasgupta, A., Majumdar, A., Ralston, R., Roy, R. & Gupta, N. K. (1978) Proc. Natl. Acad. Sci. USA 75, 745-749], we describe here the isolation and some characteristics of a factor that is required for the HRI-catalyzed inhibition of eIF-2-promoted ternary complex formation. In the presence of 1 mM Mg(2+), ternary complex formation by eIF-2 is dependent on the presence of this stabilization factor (SF). Under these conditions, SF increases the rate and the extent of ternary complex formation. This finding suggests that the interaction of SF with eIF-2 causes a conformational change that stabilizes eIF-2 and promotes efficient ternary complex formation by increasing the affinity of eIF-2 for GTP and Met-tRNA(f). In the absence of Mg(2+), however, eIF-2 efficiently forms the ternary complex and SF has little effect on its ternary complex formation capacity-hence, the name eIF-2 stabilization factor (SF). In the presence of SF, HRI markedly inhibits (70-80%) the ternary complex formation capacity of eIF-2. The inhibitory effect requires both HRI and ATP. Under these conditions, HRI phosphorylates only the 38,000-dalton subunit of eIF-2. Both the rate and the extent of the SF-dependent ternary complex formation are inhibited. These findings are consistent with the idea that phosphorylation causes a conformational change in eIF-2 such that its interactions with other initiation factors in the formation and the binding of ternary complex to 40S ribosomal subunits are inhibited.     

Tranexamic acid, an inhibitor of plasminogen activation, reduces urinary collagen cross-link excretion in both experimental and rheumatoid arthritis

The plasminogen activation system is one of the enzyme systems held responsible for bone and cartilage degradation in rheumatoid arthritis (RA). In this study, we evaluated the effect of tranexamic acid (TEA), an inhibitor of plasminogen activation, on urinary collagen cross-link excretion and radiological joint damage in rat adjuvant arthritis (AA) and on urinary collagen cross-link excretion in patients with RA. In the animal study, adjuvant arthritis was induced in male Lewis rats. From day 7 onward, high-dose TEA (500 mg/kg body weight, once daily) or placebo was administered orally. Study groups consisted of TEA-treated normal rats (C + TEA), placebo-treated normal rats (C + plac), AA rats treated with TEA (AA + TEA) or with placebo (AA + plac). To monitor joint destruction, urinary collagen cross-link excretion (pyridinoline, HP; deoxypyridinoline, LP) was measured by high-performance liquid chromatography at days 14 and 21. Radiological evaluation of joints was performed at day 21. In the patient study, TEA was administered to nine patients with RA as adjuvant medication (approximately 20 mg/kg body weight, three times daily) for 12 weeks. Urinary HP and LP excretion levels were measured before and during TEA treatment, and 4 weeks after the cessation of TEA treatment. In AA + TEA rats, a significant reduction of HP and a tendency towards a reduction of LP excretion were found compared with AA + plac rats (P < 0.05), at day 14, whereas the HP/LP ratio did not change. No difference was observed in HP, LP excretion, HP/LP ratio and radiological damage score between the TEA- and placebo-treated AA rats at day 21. In RA patients, a significant reduction of HP and LP excretion was found during the TEA treatment period (P < 0.05). After the cessation of TEA treatment, HP and LP excretion increased towards baseline levels. No effect on disease activity was observed. The plasmin antagonist TEA reduced the excretion of collagen pyridinoline cross-links in both experimental and rheumatoid arthritis. As such, this study not only supports the involvement of the plasminogen activation system in the destructive phase of arthritis, but also suggests a beneficial effect of therapeutic strategies directed against inhibition of matrix proteolysis.      

Regulation of protein synthesis in rabbit reticulocyte lysates: purification and initial characterization of the cyclic 3'':5''-AMP independent protein kinase of the heme-regulated translational inhibitor.

The heme-regulated translational inhibitor (HRI) has been purified 4800-fold. On electrophoresis in sodium dodecyl sulfate/polyacrylamide gel, the purified HRI showed one major polypeptide band. The purified HRI inhibits protein synthesis in lysates containing optimal levels of hemin with inhibition kinetics which parallel those observed in heme-deficiency. Data are presented which are consistent with an enzymatic function of HRI in the inhibition of protein synthesis. The HRI is an adenosine 3':5'-cyclic monophosphate independent protein kinase which phosphorylates the small subunit (38,000) but not the large subunits (52,000 and 50,000) of the initiation factor which forms a ternary complex with Met-tRNAf and GTP. This evidence supports the hypothesis that inhibition of protein synthesis by HRI involves the phosphorylation of the initiation factor. These findings are discussed in relation to various models for the regulation of protein kinase activity by heme. (see article).     

Somatic hypermutation in low-grade mucosa-associated lymphoid tissue- type B-cell lymphoma

The origin of low-grade mucosa-associated lymphoid tissue (MALT)-type B- cell lymphoma is still unclear. Using a novel two-step procedure, we have sequenced the Ig VH genes expressed by cells from four patients with gastric low-grade MALT-type lymphoma. The nucleotide sequences of the complementarity determining region 3 (CDR3) of the genomic DNA were first amplified using consensus oligonucleotide primers, then sequenced. Based on the CDR3 sequence amplified from each MALT lymphoma, individual tumor-specific primers were synthesized and used directly in the polymerase chain reaction (PCR) to analyze the sequences of their Ig heavy-chain variable region. When compared with the germ-line sequence, many nucleotide substitutions, mainly in the CDRs, were found in the variable gene sequences of the four MALT lymphomas. The mutations showed a high replacement-to-silent ratio and were distributed in a way which suggested that the tumor cells had been positively selected through their antigen receptor. Our findings indicate that the MALT-type lymphoma B cells are hypermutated postgerminal center lymphocytes that have undergone antigen selection.     

Deficiency of platelet membrane glycoprotein Ia associated with a decreased platelet adhesion to subendothelium: a defect in platelet spreading

A bleeding disorder with absent collagen-induced platelet aggregation and adhesion has been described in a patient whose platelets failed to express surface glycoprotein Ia. We studied the interaction of her platelets with subendothelium in an annular perfusion chamber and the interaction with purified human collagen type III in a rectangular perfusion system under flow conditions. Platelet adherence was almost completely absent both at low and high shear rates. The few platelets which adhered remained in the contact stage without subsequent spreading and aggregate formation. Addition of a monoclonal antibody, which was directed against the von Willebrand moiety of FVIII-VWF, to the blood, completely abolished platelet adherence at high shear rates and had a partial effect at low shear rates. These data indicate that von Willebrand factor plays a role in the initial attachment (contact stage) of platelets to subendothelium. We conclude that the bleeding disorder and excessively prolonged bleeding time in our patient are caused by a new specific defect of the platelet-vessel wall interaction.     

Cytomegalovirus-induced necrotizing and crescentic glomerulonephritis in a renal transplant patient

A 35-year-old black man with end-stage renal disease from biopsy-proven focal segmental glomerulosclerosis developed worsening function of his renal allograft 160 days after living related donor renal transplantation. Renal biopsy showed necrotizing and crescentic glomerulonephritis (NCGN) and presence of intraglomerular viral inclusions confirmed by immunocytochemical stain and in situ hybridization techniques to be cytomegaloviral in origin. Electron microscopy showed no immune complexes, and workup for other causes of NCGN was negative. The patient was treated with ganciclovir without other changes in his immunosuppressive regimen. After 8 weeks of ganciclovir therapy, a second renal transplant biopsy showed resolution of the glomerular process and disappearance of the cytomegalovirus (CMV) inclusions. The resolution of the glomerular process with treatment for CMV infection, and without other change in therapy, strongly supports a causative link between CMV and NCGN in this patient. This case represents the first report of CMV-associated NCGN in a renal transplant patient.