Effects of protein restriction during gestation and lactation on cell proliferation in the hippocampus and subventricular zone: Functional implications. Protein restriction alters hippocampal/SVZ cell proliferation

There is no consensus about the effects of protein restriction on neurogenesis and behavior. Here, for the first time, we evaluated the effects of protein restriction during gestation and lactation, on the two major neurogenic regions of the adult brain, the subgranular zone (SGZ) of the hippocampal dentate gyrus and the subventricular zone (SVZ), simultaneously. We also assessed different types of behavior relevant to each region. After mating, pregnant Wistar rats were divided into a control group (CG) that received a normal diet (20% protein); and a protein-restriction group (PRG) that received a low-protein diet (8% protein). After birth, the same diets were provided to the mother and pups until weaning, when some rats were analyzed and others received a normal-protein diet until adulthood. Different sets of rats were used for cellular and behavioral studies in juvenile or adult age. Brains were processed for immunohistochemistry anti-BrdU, anti-Ki67, or anti-pHisH3. Juvenile and adult rats from distinct litters also underwent several behavioral tests. Our data show that early protein restriction results in a reduction of hippocampal progenitors and deficits in object recognition during adult life. Moreover, longer periods of immobility in the tail suspension and in the forced swimming tests revealed that PRG rats show a depressive behavior at 21 days of age (P21) and in adulthood. Furthermore, we suggest that despite the reduced number/proliferation of neural stem cells (B and/or E cells) in SVZ there is a compensatory mechanism in which the progenitors (types C and A cells) proliferate in a higher rate, without affecting olfactory ability in adulthood. ► Effects of early protein restriction on cell proliferation in neurogenic niches. ► Impaired progenitor hippocampal proliferation and recognition memory, depression. ► Reduced proliferation of neural progenitor in adult SVZ without olfactory deficits

Bone marrow mesenchymal cells improve muscle function in a skeletal muscle re-injury model.

Skeletal muscle injury is the most common problem in orthopedic and sports medicine, and severe injury leads to fibrosis and muscle dysfunction. Conventional treatment for successive muscle injury is currently controversial, although new therapies, like cell therapy, seem to be promise. We developed a model of successive injuries in rat to evaluate the therapeutic potential of bone marrow mesenchymal cells (BMMC) injected directly into the injured muscle. Functional and histological assays were performed 14 and 28 days after the injury protocol by isometric tension recording and picrosirius/Hematoxilin & Eosin staining, respectively. We also evaluated the presence and the fate of BMMC on treated muscles; and muscle fiber regeneration. BMMC treatment increased maximal skeletal muscle contraction 14 and 28 days after muscle injury compared to non-treated group (4.5 ± 1.7 vs 2.5 ± 0.98 N/cm2, p<0.05 and 8.4 ± 2.3 vs. 5.7 ± 1.3 N/cm2, p<0.05 respectively). Furthermore, BMMC treatment increased muscle fiber cross-sectional area and the presence of mature muscle fiber 28 days after muscle injury. However, there was no difference in collagen deposition between groups. Immunoassays for cytoskeleton markers of skeletal and smooth muscle cells revealed an apparent integration of the BMMC within the muscle. These data suggest that BMMC transplantation accelerates and improves muscle function recovery in our extensive muscle re-injury model

Bone marrow mesenchymal cells improve muscle function in a skeletal muscle re-injury model

Skeletal muscle injury is the most common problem in orthopedic and sports medicine, and severe injury leads to fibrosis and muscle dysfunction. Conventional treatment for successive muscle injury is currently controversial, although new therapies, like cell therapy, seem to be promise. We developed a model of successive injuries in rat to evaluate the therapeutic potential of bone marrow mesenchymal cells (BMMC) injected directly into the injured muscle. Functional and histological assays were performed 14 and 28 days after the injury protocol by isometric tension recording and picrosirius/Hematoxilin & Eosin staining, respectively. We also evaluated the presence and the fate of BMMC on treated muscles; and muscle fiber regeneration. BMMC treatment increased maximal skeletal muscle contraction 14 and 28 days after muscle injury compared to non-treated group (4.5 ± 1.7 vs 2.5 ± 0.98 N/cm2, p<0.05 and 8.4 ± 2.3 vs. 5.7 ± 1.3 N/cm2, p<0.05 respectively). Furthermore, BMMC treatment increased muscle fiber cross-sectional area and the presence of mature muscle fiber 28 days after muscle injury. However, there was no difference in collagen deposition between groups. Immunoassays for cytoskeleton markers of skeletal and smooth muscle cells revealed an apparent integration of the BMMC within the muscle. These data suggest that BMMC transplantation accelerates and improves muscle function recovery in our extensive muscle re-injury model

Quantification of collagen content.

<p>Quantification of collagen content in longitudinal sections by picrosirius staining in BMMC-treated and non-treated groups soleus muscle 14 and 28 days after injury. Normal group values are contralateral non-injured muscle. Values are means ± SD of collagen area/total area ratio.</p><p>*** p<0,001 compared to normal.</p><p>Quantification of collagen content.</p

Immunolocalization of smooth muscle myosin as vessel marker in soleus muscle treated with BMMC 28 days after repeated injuries.

<p>A) and (D) Differential interference contrast images; (B) and (E) overlay images of Hoestch (in blue) and smooth muscle myosin (green) staining; (C) and (F) smooth muscle myosin alone. Green fluorescence indicates typical vessel shape labelling pattern while nuclei are shown by blue Hoescht staining. Arrows denote nuclei position. Bars correspond to 20μm.</p

Skeletal muscle troponin immunolocalization in soleus muscle treated with BMMC 28 days after repeated injuries.

<p>(A) and (D) Differential interference contrast images; (B) and (E) overlay images of Hoestch (in blue) and troponin I (green); (C) and (F) troponin I staining alone. Green fluorescence indicates typical striated skeletal muscle labelling pattern while nuclei are shown by blue Hoescht staining. Arrows denote nuclei position. Bars correspond to 20μm.</p

Histological sections stained by heamatoxylin & eosin.

<p>(A1) H&E staining image of a rat non-injured soleus muscle 28 days after surgery; (A2) same as in A1 except that vehicle-treated muscle is shown; (A3) same as in A1 but BMMC-treated animal is presented; (A2, A3 and A4) Details of A1, A2 and A3 animals are shown in greater magnification, respectively; (A3) Picrossírius staining image of a rat non-injured soleus muscle 28 days after surgery;; (B3) same as in A3 except that vehicle-treated muscle is shown; (C3) same as in A3 but BMMC-treated animal is presented and (A4, B4 and C4) Details of A3, B3 and C3 animals are shown in greater magnification, respectively; Histological analysis reveals that both groups submitted to muscle injury present wide spread collagen deposition (A3-C4) independent of treatment. Note the central aligned disposition of the muscle fiber nuclei indicating recent muscle fiber regeneration (arrows in B2). Bars correspond to 300 μm in A1-C1 and A3-C3 and correspond to 50μm in A2-C2 and A4-C4.</p