Micro-computed tomography(micro-CT) is becoming an accepted research tool for the noninvasive examination of laboratory animals such as mice and rats, but to date, in vivo scanning has largely been limited to the evaluation of skeletal tissues. We use a commercially available micro-CT device to perform respiratory gated in vivo acquisitions suitable for thoracic imaging. The instrument is described, along with the scan protocol and animal preparation techniques. Preliminary results confirm that lung tumors as small as 1 mm in diameter are visible in vivo with these methods. Radiation dose was evaluated using several approaches, and was found to be approximately 0.15 Gy for this respiratory-gated micro-CT imaging protocol. The combination of high-resolution CT imaging and respiratory-gated acquisitions appears well-suited to serial in vivo scanning.

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/b-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 standalone therapy or as part of a combination therapeutic strategy for the treatment of TBI. STEM CELLS 2016;34:1263-1272 SIGNIFICANCE STATEMENTMesenchymal stem cells (MSCs) have been shown to have therapeutic potential in treating a number of diseases, including traumatic brain injury (TBI), yet their exact mechanisms of action are not well-understood. Here we show that IV-MSCs protect newborn hippocampal neurons and promote neurogenesis in traumatic brain injured mice. We identified Wnt3a as a soluble factor, released systemically after IV MSC administration that is involved in the neuroprotective and neurogenic effects of IV-MSCs. In support of this premise, we find that IV administration of Wnt3a post-TBI attenuates the loss of neural stem cells, enhances neurogenesis, and improves neuro cognitive function. These results are novel, have not been previously reported, and support the clinical investigation of IV-Wnt3a as a treatment for TBI.

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. STEM CELLS 2015;33:3530-3544 SIGNIFICANCE STATEMENTTBI is the main cause of death worldwide in ages 1-44 with few if any effective therapeutic options to treat patients. Our data suggest that TIMP3, a protein released by Mesenchymal Stem Cells may be a worthy therapeutic candidate. We report that TIMP3 has direct and potent effects on neuronal survival and neurocognitive function post TBI. This study builds upon our previous work that showed TIMP3 is protective against blood brain barrier compromise in the disease. Our findings report a critical role of the Akt-mTORC1 pathway and that inhibition of this pathway abrogates the neuroprotective effect of TIMP3.

Micro-computed tomography (micro-CT) is becoming an accepted research tool for the noninvasive examination of laboratory animals such as mice and rats, but to date, in vivo scanning has largely been limited to the evaluation of skeletal tissues. We use a commercially available micro-CT device to perform respiratory gated in vivo acquisitions suitable for thoracic imaging. The instrument is described, along with the scan protocol and animal preparation techniques. Preliminary results confirm that lung tumors as small as 1 mm in diameter are visible in vivo with these methods. Radiation dose was evaluated using several approaches, and was found to be approximately 0.15 Gy for this respiratory-gated micro-CT imaging protocol. The combination of high-resolution CT imaging and respiratory-gated acquisitions appears well-suited to serial in vivo scanning. Mol Imaging (2004) 3, 55-62.

No abstract