A multi-center study on low-frequency rTMS combined with intensive occupational therapy for upper limb hemiparesis in post-stroke patients

<p>Abstract</p> <p>Background</p> <p>Both low-frequency repetitive transcranial magnetic stimulation (rTMS) and intensive occupational therapy (OT) have been recently reported to be clinically beneficial for post-stroke patients with upper limb hemiparesis. Based on these reports, we developed an inpatient combination protocol of these two modalities for the treatment of such patients. The aims of this pilot study were to confirm the safety and feasibility of the protocol in a large number of patients from different institutions, and identify predictors of the clinical response to the treatment.</p> <p>Methods</p> <p>The study subjects were 204 post-stroke patients with upper limb hemiparesis (mean age at admission 58.5 ± 13.4 years, mean time after stroke 5.0 ± 4.5 years, ± SD) from five institutions in Japan. During 15-day hospitalization, each patient received 22 treatment sessions of 20-min low-frequency rTMS and 120-min intensive OT daily. Low-frequency rTMS of 1 Hz was applied to the contralesional hemisphere over the primary motor area. The intensive OT, consisting of 60-min one-to-one training and 60-min self-exercise, was provided after the application of low-frequency rTMS. Fugl-Meyer Assessment (FMA) and Wolf Motor Function Test (WMFT) were performed serially. The physiatrists and occupational therapists involved in this study received training prior to the study to standardize the therapeutic protocol.</p> <p>Results</p> <p>All patients completed the protocol without any adverse effects. The FMA score increased and WMFT log performance time decreased significantly at discharge, relative to the respective values at admission (change in FMA score: median at admission, 47 points; median at discharge, 51 points; p < 0.001. change in WMFT log performance time: median at admission, 3.23; median at discharge, 2.51; p < 0.001). These changes were persistently seen up to 4 weeks after discharge in 79 patients. Linear regression analysis found no significant relationship between baseline parameters and indexes of improvement in motor function.</p> <p>Conclusions</p> <p>The 15-day inpatient rTMS plus OT protocol is a safe, feasible, and clinically useful neurorehabilitative intervention for post-stroke patients with upper limb hemiparesis. The response to the treatment was not influenced by age or time after stroke onset. The efficacy of the intervention should be confirmed in a randomized controlled study including a control group.</p

Adipose-Derived Mesenchymal Stem Cells Restore Impaired Mucosal Immune Responses in Aged Mice

<div><p>It has been shown that adipose-derived mesenchymal stem cells (AMSCs) can differentiate into adipocytes, chondrocytes and osteoblasts. Several clinical trials have shown the ability of AMSCs to regenerate these differentiated cell types. Age-associated dysregulation of the gastrointestinal (GI) immune system has been well documented. Our previous studies showed that impaired mucosal immunity in the GI tract occurs earlier during agingthan is seen in the systemic compartment. In this study, we examined the potential of AMSCs to restore the GI mucosal immune system in aged mice. Aged (>18 mo old) mice were adoptively transferred with AMSCs. Two weeks later, mice were orally immunized with ovalbumin (OVA) plus cholera toxin (CT) three times at weekly intervals. Seven days after the final immunization, when fecal extract samples and plasma were subjected to OVA- and CT-B-specific ELISA, elevated levels of mucosal secretory IgA (SIgA) and plasma IgG antibody (Ab) responses were noted in aged mouse recipients. Similar results were also seen aged mice which received AMSCs at one year of age. When cytokine production was examined, OVA-stimulated Peyer’s patch CD4⁺ T cells produced increased levels of IL-4. Further, CD4⁺ T cells from the lamina propria revealed elevated levels of IL-4 and IFN-γ production. In contrast, aged mice without AMSC transfer showed essentially no OVA- or CT-B-specific mucosal SIgA or plasma IgG Ab or cytokine responses. Of importance, fecal extracts from AMSC transferred aged mice showed neutralization activity to CT intoxication. These results suggest that AMSCs can restore impaired mucosal immunity in the GI tract of aged mice.</p></div

OVA-specific Ab responses in aged and young adult mice.

<p>Aged mice were adoptively transferred with mAMSCs as described in the Materials And Methods section. Aged mice given mAMSCs (hatched bar), naïve aged mice (without mAMSC transfer, open bar) and young adult mice (closed bar) were orally immunized with 1 mg OVA plus 10 μg of CT three times at weekly intervals. One week after the last immunization, levels of anti-OVA fecal SIgA and plasma IgG Abs were determined by OVA-specific ELISA. The values shown are the mean ± SEM taken from 10 mice in each group. *<i>p</i> < 0.05 when compared with aged mice without AMSC adoptive transfer.</p

Functional properties of CT-B-specific SIgA Abs in aged mice adoptively transferred with AMSCs.

<p>Each fecal extract sample (200 μl) from (A and F) naïve aged mice, (B) aged mice given oral OVA plus CT, (C) AMSC adoptively transferred aged mice given oral OVA plus CT, (D) naïve young adult mice or (E) young adult mice given oral OVA plus CT, was injected into the ligation of jejunum loops (4–6 cm) of mice together with 20 μl of CT (1 mg/ml]) (A-E) or (F) PBS. Twelve hours after injection, each loop was hung on a fixed clip and stretched by placing another clip on the other end of the loop. The length and weight of each loop were measured. The volume/length ratio (μl / cm) was used to express the intensity of the reaction. The results represent the mean values ± SEM of 6 mice in each experimental group. *<i>p</i> < 0.05 when compared with aged mice without mAMSC adoptive transfer or naïve aged mice. The pictures represent typical results and are taken from one of three separate experiments.</p

Th1- and Th2-type cytokine production by CD4⁺ T cells.

<p>The CD4⁺ T cells (4 x 10⁶ cells/ml) from aged mice given oral OVA plus CT with (closed bars) or without (open bars) mAMSC transfer were purified from PPs, LP, and spleen one week after the final immunization. Cells were then cultured with 1 mg/ml OVA in the presence of irradiated splenic antigen-presenting cells (8 x 10⁶ cells/ml). Culture supernatants were harvested after 5 days of incubation and analyzed by the respective cytokine-specific ELISA. The levels of Ag-stimulated cytokine production were calculated by subtraction of the results of culture without OVA stimulation from those with OVA stimulation. The values shown are the mean ± SEM of 10 mice in each experimental group. *<i>p</i> < 0.05 when compared with aged mice without AMSC transfer.</p

CT-B-specific Ab responses in aged and young adult mice.

<p>Aged mice were adoptively transferred with mAMSCs or hAMSCs. Aged mice given mAMSCs (hatched bar), naïve aged mice (without hAMSC transfer, open bar) and young adult mice (closed bar) were orally immunized with 1 mg of OVA plus 10 μg of CT three times at weekly intervals. One week after the last immunization, levels of anti-CT-B fecal SIgA and plasma IgG Abs were determined by CT-B-specific ELISA. The values shown are the mean ± SEM taken from 10 mice in each group. *<i>p</i> < 0.05 when compared with aged mice without either mAMSC or hAMSC transfer.</p

OVA-specific AFCs in lamina propria (LP) and spleen of aged mice.

<p>Aged mice with (○) or without (□) mAMSC transfer were orally immunized as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148185#pone.0148185.g001" target="_blank">Fig 1</a> legend. One week after the last immunization, mononuclear cells were isolated from the LP and spleen and were then subjected to an OVA-specific ELISPOT assay to determine the numbers of IgA and IgG AFCs. Naïve mice served as a control group and did not exhibit any anti-OVA AFCs (data not shown). The values shown are the mean ± SEM taken from 10 mice in each group. *<i>p</i> < 0.05 when compared with aged mice without adoptive transfer of mAMSCs.</p

OVA-specific Ab responses in aged mice given human AMSCs (hAMSCs).

<p>Aged mice were adoptively transferred with hAMSCs. Aged mice adoptively transferred with hAMSCs (hatched bar), naïve aged mice (without hAMSC transfer, open bar) and young adult mice (closed bar) were orally immunized with 1 mg of OVA plus 10 μg CT three times at weekly intervals. One week after the last immunization, levels of anti-OVA fecal SIgA and plasma IgG Abs were determined by an OVA-specific ELISA. The values shown are the mean ± SEM taken from 10 mice in each group. *<i>p</i> < 0.05 when compared with aged mice without AMSC adoptive transfer.</p

Interleukin-4 and IFN-γ production by CD4⁺ T cells from PPs, LP and spleen were determined by intracellular analysis.

<p>Mononuclear cells were isolated from aged mice given oral OVA plus CT with or without mAMSC adoptive transfer. Cells were incubated with ionomycin (1 μg/ml) and phorbol 12-myristate 13-acetate (PMA; 25 ng/ml) for 3 hr and then stained with FITC-labeled anti-CD4 mAb. Samples were further stained intracellularly with PE-labeled anti-IL-4 (A) or anti-IFN-γ (B) mAb. The frequencies of cytokine-producing cells were determined by flow cytometry. The profiles represent typical results and are taken from one of three separate experiments.</p