BackgroundTraumatic brain injury (TBI) is a major cause for long-term disability, yet the treatments available that improve outcomes after TBI limited. Neuroinflammatory responses are key contributors to determining patient outcomes after TBI. Transplantation of mesenchymal stem cells (MSCs), which release trophic and pro-repair cytokines, represents an effective strategy to reduce inflammation after TBI. One such pro-repair cytokine is interleukin-10 (IL-10), which reduces pro-inflammatory markers and trigger alternative inflammatory markers, such as CD163. In this study, we tested the therapeutic effects of MSCs that were engineered to overexpress IL-10 when transplanted into rats following TBI in the medial frontal cortex.MethodsThirty-six hours following TBI, rats were transplanted with MSCs and then assessed for 3 weeks on a battery of behavioral tests that measured motor and cognitive abilities. Histological evaluation was then done to measure the activation of the inflammatory response. Additionally, immunomodulatory effects were evaluated by immunohistochemistry and Western blot analyses.ResultsA significant improvement in fine motor function was observed in rats that received transplants of MSCs engineered to overexpress IL-10 (MSCs + IL-10) or MSCs alone compared to TBI + vehicle-treated rats. Although tissue spared was unchanged, anti-inflammatory effects were revealed by a reduction in the number of glial fibrillary acidic protein cells and CD86 cells in both TBI + MSCs + IL-10 and TBI + MSC groups compared to TBI + vehicle rats. Microglial activation was significantly increased in the TBI + MSC group when compared to the sham + vehicle group. Western blot data suggested a reduction in tumor necrosis factor-alpha in the TBI + MSCs + IL-10 group compared to TBI + MSC group. Immunomodulatory effects were demonstrated by a shift from classical inflammation expression (CD86) to an alternative inflammation state (CD163) in both treatments with MSCs and MSCs + IL-10. Furthermore, co-labeling of both CD86 and CD163 was detected in the same cells, suggesting a temporal change in macrophage expression.ConclusionsOverall, our findings suggest that transplantation of MSCs that were engineered to overexpress IL-10 can improve functional outcomes by providing a beneficial perilesion environment. This improvement may be explained by the shifting of macrophage expression to a more pro-repair state, thereby providing a possible new therapy for treating TBI.
Drug delivery into the central nervous system (CNS) is challenging due to the blood-brain barrier (BBB) and drug delivery into the brain overcoming the BBB can be achieved using nanoparticles such as dendrimers. The conventional cationic dendrimers used are highly toxic. Therefore, the present study investigates the role of novel mixed surface dendrimers, which have potentially less toxicity and can cross the BBB when administered through the carotid artery in mice. In vitro experiments investigated the uptake of amine dendrimers (G1-NH 2 and G4-NH 2 ) and novel dendrimers (G1-90/10 and G4-90/10) by primary cortical cultures. In vivo experiments involved transplantation of G4-90/10 into mice through (1) invasive intracranial injections into the striatum; and (2) less invasive carotid injections. The animals were sacrificed 24-h and 1-week post-transplantations and their brains were analyzed. In vivo experiments proved that the G4-90/10 can cross the BBB when injected through the carotid artery and localize within neurons and glial cells. The dendrimers were found to migrate through the corpus callosum 1-week post intracranial injection. Immunohistochemistry showed that the migrating cells are the dendrimer-infected glial cells. Overall, our results suggest that poly-amidoamine (PAMAM) dendrimers may be used as a minimally invasive means to deliver biomolecules for treating neurological diseases or disorders Keywords: PAMAM dendrimer nanoparticle; blood-brain barrier; non-invasive delivery; bio-distribution and uptake; neurodegenerative diseases Int.
Autophagy, including mitophagy, is critical for neuroprotection in traumatic brain injury (TBI). Transplantation of mesenchymal stem cells (MSCs) provides neuroprotection and induces autophagy by increasing anti‐inflammatory cytokines, such as interleukin‐10 (IL‐10). To evaluate these effects of IL10 that are released by MSCs, we genetically engineered MSCs to overexpress IL10 and compared their effects to unaltered MSCs following transplantation near the site of induced TBIs in rats. Adult, male Sprague‐Dawley rats were divided into four groups: Sham + vehicle, TBI + vehicle, TBI + MSCs‐IL‐10 and TBI + MSCs‐GFP. Thirty‐six hours post‐TBI, the first two groups received vehicle (Hanks balance salt solution), whereas last two groups were transplanted with MSCs‐IL‐10 or MSCs‐GFP. Three weeks after transplantation, biomarkers for neurodegenerative changes, autophagy, mitophagy, cell death and survival markers were measured. We observed a significant increase in the number of dead cells in the cortex and hippocampus in TBI rats, whereas transplantation of MSCs‐IL‐10 significantly reduced their numbers in comparison to MSCs alone. MSCs‐IL‐10 rats had increased autophagy, mitophagy and cell survival markers, along with decreased markers for cell death and neuroinflammation. These results suggest that transplantation of MSCs‐IL‐10 may be an effective strategy to protect against TBI‐induced neuronal damage.
Drug delivery to the brain is highly hindered by the presence of the blood–brain barrier (BBB), which prevents the entry of many potential drugs/biomolecules into the brain. One of the current strategies to achieve gene therapy for neurodegenerative diseases involves direct injection of a viral vector into the brain. There are various disadvantages of viral vectors, including limitations of cargo size and safety concerns. Nanomolecules, such as dendrimers, serve as an excellent alternative to viral delivery. In this study, as proof-of-concept, we used a surface-modified dendrimer complex and delivered large plasmids to cells in vitro and in vivo in healthy rats via intracranial injection. The dendrimers were biodegradable by chemicals found within cells and toxicity assays revealed that the modified dendrimers were much less toxic than unmodified amine-surface dendrimers. As mentioned in our previous publication, these dendrimers with appropriately modified surfaces are safe, can deliver large plasmids to the brain, and can overcome the cargo size limitations associated with viral vectors. The biocompatibility of this dendritic nanomolecule and the ability to finely tune its surface chemistry provides a gene delivery system that could facilitate future in vivo cellular reprograming and other gene therapies.
A major drawback of current stroke treatment strategies (such as the use of tPA) includes time sensitivity to achieve maximum therapeutic efficacy. Alternative treatments include less time-sensitive approaches and utilize
in vivo
reprogramming of resident reactive astrocytes to repopulate the lost neurons in sufficient numbers. In this study, we tested whether a transcription factor,
hSOX2,
when expressed under a glial cell-specific GFAP promoter, could sufficiently reprogram astrocytes in and around the infarct to enhance their differentiation into neurons. To achieve delivery of the hSOX2 gene, we utilize PAMAM dendrimers, which are nanomolecules with a well-established capacity of delivering drugs/ large biomolecules to the brain across the BBB and confer intrinsic anti-inflammatory properties. Dendrimers are comprised of an interior dendritic structure with modifiable sizes, and an exterior surface with functional surface groups. The G4 PAMAM dendrimers used in this study has 10% of the surface covered with amine groups and 90% of the surface covered with hydroxyl groups (G4-90/10). These dendrimers are less toxic and readily form complexes with plasmids up to 14 kb in size (dendriplex) and successfully deliver cargo
in vitro
and
in vivo
. Four days following stroke inductions in Sprague Dawley rats via MCAo, the hSOX2 dendriplex was injected into the ipsilateral corpus callosum. A battery of behavior tests, such as cylinder and ladder tasks, were used to assess motor abilities of the treated and untreated stroked and sham-operated control rats. Moreover, the animals underwent In vivo Imaging System to confirm the presence of dendriplex in the brain. Five weeks following the injections, the brains were collected and processed, using immunohistochemistry, to detect the complex and measure the amount of
hSOX2
gene expression. The size of the brain infarct was measured using the conventional H&E staining. Our results indicated that the dendrimers were able to deliver the hSOX2 gene to the stroke brain and this significantly reduced motor deficits, relative to untreated stroked rats. These results indicated that PAMAM dendrimers effectively deliver genes into the brain and that the hSOX2 gene can successfully reduce motor deficits following stroke.
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