Wnt3a, a Protein Secreted by Mesenchymal Stem Cells Is Neuroprotective and Promotes Neurocognitive Recovery Following Traumatic Brain Injury.

Intravenous administration of bone marrow derived mesenchymal stem cells (MSCs) has been shown to reduce blood brain barrier compromise and improve neurocognition following traumatic brain injury (TBI). These effects occur in the absence of engraftment and differentiation of these cells in the injured brain. Recent studies have shown that soluble factors produced by MSCs mediate a number of the therapeutic effects. In this study, we sought to determine if intravenous administration of MSCs (IV-MSCs) could enhance hippocampal neurogenesis following TBI. Our results demonstrate that IV-MSC treatment attenuates loss of neural stem cells and promotes hippocampal neurogenesis in TBI injured mice. As Wnt signaling has been implicated in neurogenesis, we measured circulating Wnt3a levels in serum following IV-MSC administration and found a significant increase in Wnt3a. Concurrent with this increase, we detected increased activation of the Wnt/ß-catenin signaling pathway in hippocampal neurons. Furthermore, IV recombinant Wnt3a treatment provided neuroprotection, promoted neurogenesis, and improved neurocognitive function in TBI injured mice. Taken together, our results demonstrate a role for Wnt3a in the therapeutic potential of MSCs and identify Wnt3a as a potential stand-alone therapy or as part of a combination therapeutic strategy for the treatment of TBI. Stem Cells 2016;34:1263-1272.

TIMP3 Attenuates the Loss of Neural Stem Cells, Mature Neurons and Neurocognitive Dysfunction in Traumatic Brain Injury.

Mesenchymal stem cells (MSCs) have been shown to have potent therapeutic effects in a number of disorders including traumatic brain injury (TBI). However, the molecular mechanism(s) underlying these protective effects are largely unknown. Herein we demonstrate that tissue inhibitor of matrix metalloproteinase-3 (TIMP3), a soluble protein released by MSCs, is neuroprotective and enhances neuronal survival and neurite outgrowth in vitro. In vivo in a murine model of TBI, intravenous recombinant TIMP3 enhances dendritic outgrowth and abrogates loss of hippocampal neural stem cells and mature neurons. Mechanistically we demonstrate in vitro and in vivo that TIMP3-mediated neuroprotection is critically dependent on activation of the Akt-mTORC1 pathway. In support of the neuroprotective effect of TIMP3, we find that intravenous delivery of recombinant TIMP3 attenuates deficits in hippocampal-dependent neurocognition. Taken together, our data strongly suggest that TIMP3 has direct neuroprotective effects that can mitigate the deleterious effects associated with TBI, an area with few if any therapeutic options.

Flat-panel versus 64-channel computed tomography for in vivo quantitative characterization of aortic atherosclerotic plaques.

BACKGROUND: Flat-panel computed tomography (FpCT) provides better spatial resolution than 64-channel CT (64-CT) and may improve in vivo quantitative assessment of atherosclerotic plaques. METHODS AND RESULTS: Lesions in 184 aortic histology sections from 6 Watanabe heritable hyperlipidemic rabbits were quantitatively compared with 64-CT (image thickness, 0.625 mm) and FpCT (image thickness, 0.150 mm) images. Images were re-oriented perpendicular to the vessel centerline. For detecting plaque, FpCT and 64-CT were not significantly different (sensitivity, 76% vs 66%; P=NS). Although FpCT was significantly more sensitive (42% vs 0%; P=<0.001) for detecting eccentric lesions, the area under the curve (AUC) for FpCT (0.6) was not significantly different from that for 64-CT (0.45; P=NS). In detecting plaques with ≤ 10% lipid (low attenuation foci), FpCT was significantly more sensitive than 64-CT (24% vs 0.7%; P<0.00) and had a significantly greater AUC (0.6 vs 0.5; P<0.006). Additionally, FpCT was more sensitive (65% vs 0%; P<0.00) in detecting plaques with ≤ 5% calcium (high attenuation foci) but not in detecting branch points. Both FpCT and histology allowed us to detect low-attenuation foci as small as 0.3mm in diameter, whereas 64-CT allowed us to detect only low-attenuation foci ≥ 1.5mm in diameter. CONCLUSIONS: Flat-panel CT seemed to have more potential for quantitatively screening low-risk small atherosclerotic lesions, whereas 64-CT was apparently more useful when imaging established, well-characterized lesions, particularly when measuring the vascular wall thickness in a rabbit model of atherosclerosis.

Quantification of mineralized bone response to prostate cancer by noninvasive in vivo microCT and non-destructive ex vivo microCT and DXA in a mouse model.

BACKGROUND: To compare nondestructive in vivo and ex vivo micro-computed tomography (muCT) and ex vivo dual-energy-X-ray-absorptiometry (DXA) in characterizing mineralized cortical and trabecular bone response to prostate cancer involving the skeleton in a mouse model. METHODOLOGY/PRINCIPAL FINDINGS: In vivo microCT was performed before and 10 weeks after implantation of human prostate cancer cells (MDA-PCa-2b) or vehicle into SCID mouse femora. After resection, femora were imaged by nondestructive ex vivo specimen microCT at three voxel sizes (31 micro, 16 micro, 8 micro) and DXA, and then sectioned for histomorphometric analysis of mineralized bone. Bone mineral density (BMD), trabecular parameters (number, TbN; separation, TbSp; thickness, TbTh) and mineralized bone volume/total bone volume (BV/TV) were compared and correlated among imaging methods and histomorphometry. Statistical tests were considered significant if P<0.05. Ten weeks post inoculation, diaphyseal BMD increased in the femur with tumor compared to the opposite femur by all modalities (p<0.005, n = 11). Diaphyseal BMD by in vivo microCT correlated with ex vivo 31 and 16 microm microCT and histomorphometry BV/TV (r = 0.91-0.94, P<0.001, n = 11). DXA BMD correlated less with bone histomorphometry (r = 0.73, P<0.001, n = 11) and DXA did not distinguish trabeculae from cortex. By in vivo and ex vivo microCT, trabecular BMD decreased (P<0.05, n = 11) as opposed to the cortex. Unlike BMD, trabecular morphologic parameters were threshold-dependent and when using "fixed-optimal-thresholds," all except TbTh demonstrated trabecular loss with tumor and correlated with histomorphometry (r = 0.73-0.90, P<0.05, n = 11). CONCLUSIONS/SIGNIFICANCE: Prostate cancer involving the skeleton can elicit a host bone response that differentially affects the cortex compared to trabeculae and that can be quantified noninvasively in vivo and nondestructively ex vivo.

Human cancellous bone from T12-L1 vertebrae has unique microstructural and trabecular shear stress properties.

Increase of trabecular stress variability with loss of bone mass has been implicated as a mechanism for increased cancellous bone fragility with age and disease. In the current study, a previous observation that trabecular shear stress estimates vary along the human spine such that the cancellous tissue from the thoracic 12 (T12)-lumbar 1 (L1) junction experiences the highest trabecular stresses for a given load was tested as a formal hypothesis using multiple human spines. Thoracic 4, T5, T7, T9, T10, T12, L1, L2, L4 and L5 vertebrae from 10 human cadaver spines were examined. One specimen in the central anterior region was cored in the supero-inferior (SI) direction and another in the postero-lateral region was cored in the transverse (TR) direction from each vertebra. Micro-CT-based large-scale finite element models were constructed for each specimen and compression in the long axis of the cylindrical specimens was simulated. Cancellous bone modulus and the mean, the standard deviation, variability and amplification of trabecular von Mises stresses were computed. Bone volume fraction, trabecular number, trabecular thickness, trabecular separation, connectivity density and degree of anisotropy were calculated using 3D stereology. The results were analyzed using a mixed model in which spine level was modeled using a quadratic polynomial. The maximum of trabecular shear stress amplification and minimum of bone volume fraction were found in the cancellous tissue from the T12-L1 location when results from the samples of the same vertebra were averaged. When groups were separated, microstructure and trabecular stresses varied with spine level, extrema being at the T12-L1 levels, for the TR specimens only. SI/TR ratio of measured parameters also had quadratic relationships with spine level, the extrema being located at T12-L1 levels for most parameters. For microstructural parameters, these ratios approached to a value of one at the T12-L1 level, suggesting that T12-L1 vertebrae have more uniform cancellous tissue properties than other levels. The mean intercept length in the secondary principal direction of trabecular orientation could account for the variation of all mechanical parameters with spine level. Our results support that cancellous tissue from T12-L1 levels is unique and may explain, in part, the higher incidence of vertebral fractures at these levels.

A new method for respiratory gating during microcomputed tomography of lung in mice.

This study investigated the use of regulated cyclic breath-holds to improve microcomputed tomography (microCT) imaging of small (diameter, less than 1 mm) mouse lung tumors in vivo. Two novel techniques that use a modified small-animal ventilator were examined and compared with a previously used respiratory gating microCT technique and a free-breathing microCT technique. Two mice were scanned with each of these 4 microCT techniques (voxel size, 92 microm). The appearance of small lung tumors (maximal diameter, 0.5 to 1.0 mm) and the characteristics of line profiles of the lung-diaphragm boundary were used to compare the images obtained from the 4 acquisition techniques. The use of cyclic breath-holds, synchronized with the CT exposures, led to marked improvement in the visualization of the mouse lung structure and lesion conspicuity. A secondary experiment was performed to assess the stress placed on mice by the acquisition techniques.

Intraperitoneal administration of an iodine-based contrast agent to improve abdominal micro-computed tomography imaging in mice.

The purpose of this study was to estimate the optimal volume of an iodine-based contrast agent to administer to mice via intraperitoneal injection and the optimal time after injection to perform micro-computed tomography for maximal enhancement of abdominal organs. Eight mice were paired randomly; three pairs underwent imaging after receiving intraperitoneal injections of 125, 250, or 500 microl of contrast agent, and the fourth pair underwent imaging without receiving an injection. Each mouse was scanned three consecutive times, and each scan lasted 25 min so that we could observe the clearance of the contrast agent from the abdomen. We determined that introducing 250 microl of contrast agent into the abdominal cavity of the mice and then having the mice undergo micro-computed tomography 15 min after injection provided the optimal degree of contrast enhancement needed to distinguish the abdominal organs. These results may lead to expanded use of this imaging modality to assess abdominal organ margins in small-animal studies in vivo.

Inhibition of prostate cancer-meditated osteoblastic bone lesions by the low-calcemic analog 1alpha-hydroxymethyl-16-ene-26,27-bishomo-25-hydroxy vitamin D3.

Prostate cancer metastasizes almost exclusively into the bone whereby it induces primarily an osteoblastic response. Non-calcemic vitamin D analogs have been shown to inhibit proliferation of prostate cancer cells in culture and inhibit their growth as subcutaneous xenografts in mice. However, their effect on prostate cancer cell growth in the bone has not been examined. In the present study, we inoculated the osteoblastic prostate cancer cell line MDA-PCa 2b into the bone of male SCID mice and examined the effect of the low-calcemic hybrid analog 1alpha-hydroxymethyl-16-ene-26,27-bishomo-25-hydroxy vitamin D(3) (JK-1626-2) on their ability to induce bone lesions. We found that 7 weeks after inoculation of MDA-PCa 2b cells, 90% of the mice in the vehicle-treated group had significant bone lesions that were detectable by micro-computed tomography and characterized by thickening of the cortical bone and ossification of the epiphysis. Only 30% of the mice in the analog-treated group (daily injections of 4microg/kg, 5 days/week for up to 7 weeks) had detectable bone lesions. Histological examination of the decalcified tumor-bearing bones has shown that tumor cells completely replaced the bone marrow in the diaphysis, and destroyed the trabecular bone in the metaphysis in 90% of the vehicle-treated mice. In contrast, the metaphysis of 60% of analog-treated mice appeared normal, although tumor cells were still found in the diaphysis of 70% of the bones in the analog-treated group. There was no evidence of hypercalcemia in any of the analog-treated mice. In a co-culture, MDA-PCa 2b cells induced a profound mitogenic response in osteoblasts followed by enhanced differentiation. However, in the presence of the analog the mitogenic response of the osteoblasts to the malignant cells was significantly attenuated. These experiments led to the hypothesis that, in vivo, JK-1626-2 prevented the metastatic bone lesions by inhibiting the mitogenic response of osteoblasts to growth factors produced by MDA-PCa 2b cells.