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Platelets (PLTs) stored at 4°C exhibit equivalent or superior hemostatic function compared with 22°C PLTs, but have shorter circulation times and a decreased ability to modulate vascular permeability. These differences may be due to morphological changes and storage-induced activation. Using a proteomics-based approach, we found that 4°C-stored PLTs express decreased α-tubulin, a key PLT structural protein. PLT activation is characterized by α-tubulin deacetylation, which is regulated by histone deacetylase-6 (HDAC-6). We hypothesized that inhibition of HDAC-6 in stored PLTs will improve their ability to regulate vascular permeability through reduced activation and α-tubulin deacetylation. In an in vivo model of vascular permeability, treatment of 4°C PLTs with the HDAC-6 inhibitor tubacin enhanced the vasculoprotective properties of untreated 4°C PLTs. 4°C PLT circulation, however, was unchanged by tubacin treatment, suggesting that circulation time may not be a critical factor in determining the vasculoprotective effects of PLTs. Assessing the factor content of stored PLTs revealed that angiopoietin-1 (Ang-1) increased in 4°C PLTs over time, which was further enhanced by tubacin treatment. In addition, angiopoietin-2, an inducer of vascular leak and antagonist of Ang-1, inhibited PLT barrier protection, suggesting involvement of the Tie-2 pathway. This study demonstrates that HDAC-6 inhibition with tubacin attenuates the diminished vasculo-protective properties of 4°C PLTs, and these properties may be independent of PLT circulation time.

BackgroundHemorrhagic shock and trauma (HS/T)-induced gut injury may play a critical role in the development of multi-organ failure. Novel therapies that target gut injury and vascular permeability early after HS/T could have substantial impacts on trauma patients. In this study, we investigate the therapeutic potential of human mesenchymal stem cells (MSCs) and MSC-derived extracellular vesicles (MSC EVs) in vivo in HS/T in mice and in vitro in Caco-2 human intestinal epithelial cells.MethodsIn vivo, using a mouse model of HS/T, vascular permeability to a 10-kDa dextran dye and histopathologic injury in the small intestine and lungs were measured among mice. Groups were (1) sham, (2) HS/T + lactated Ringer's (LR), (3) HS/T + MSCs, and (4) HS/T + MSC EVs. In vitro, Caco-2 cell monolayer integrity was evaluated by an epithelial cell impedance assay. Caco-2 cells were pretreated with control media, MSC conditioned media (CM), or MSC EVs, then challenged with hydrogen peroxide (H2O2).ResultsIn vivo, both MSCs and MSC EVs significantly reduced vascular permeability in the small intestine (fluorescence units: sham, 456 ± 88; LR, 1067 ± 295; MSC, 765 ± 258; MSC EV, 715 ± 200) and lung (sham, 297 ± 155; LR, 791 ± 331; MSC, 331 ± 172; MSC EV, 303 ± 88). Histopathologic injury in the small intestine and lung was also attenuated by MSCs and MSC EVs. In vitro, MSC CM but not MSC EVs attenuated the increased permeability among Caco-2 cell monolayers challenged with H2O2.ConclusionMesenchymal stem cell EVs recapitulate the effects of MSCs in reducing vascular permeability and injury in the small intestine and lungs in vivo, suggesting MSC EVs may be a potential cell-free therapy targeting multi-organ dysfunction in HS/T. This is the first study to demonstrate that MSC EVs improve both gut and lung injury in an animal model of HS/T.

BACKGROUND Hemorrhagic shock (HS) and trauma can result in an endotheliopathy of trauma, characterized by endothelial compromise, inflammation, and aberrant coagulation. Kcentra, a prothrombin concentrate, has been demonstrated to mitigate pulmonary vascular leak in a murine model of HS. We investigated the effects of Kcentra in a rat model of HS, to achieve physiologic endpoints of relevance. METHODS Rats subjected to a grade intravenous splenic injury and controlled hemorrhage for 60 minutes were resuscitated with shed volumes of (1) Lactated Ringer's (LR) solution, (2) LR + 20 IU/kg Kcentra, (3) LR + 50 IU/kg Kcentra, (4) rat fresh frozen plasma (RFFP), or (5) human fresh frozen plasma (HFFP). Blood was harvested for monitoring metabolic and coagulation function. Rat lungs were evaluated for lung injury and permeability. RESULTS Animals resuscitated with LR displayed a significant increase in pulmonary vascular permeability (sham, 407.9 ± 122.4; shock + LR, 2040 ± 1462). Resuscitation with RFFP (606.5 ± 169.3) reduced leak; however, treatment with Kcentra (HS + Kcentra [20 IU/kg]: 1792 ± 903.4, HS + Kcentra [50 IU/kg]: 1876 ± 1103), and HFFP (1450 ± 533.2) had no significant effect on permeability. Kcentra modestly altered clotting parameters. Metabolic measures, such as lactate, pH, and base deficit, were restored to baseline levels by both RFFP and HFFP, but not Kcentra or LR. CONCLUSION Kcentra did not alter pulmonary vascular permeability, but modestly increased clotting potential in injured rats. This suggests that there may be a xenogenic reaction of human products in rats and that the effects of Kcentra on vascular stability may be distinct from its ability to modulate clotting. Our data indicate that the species chosen and utilized for in vivo preclinical testing of human derived blood products is of critical importance in determining their efficacy in animal models and is the primary impetus to communicate these results.

Blood vessels in the central nervous system (CNS) form a specialized and critical structure, the blood-brain barrier (BBB). We present a resource to understand the molecular mechanisms that regulate BBB function in health and dysfunction during disease. Using endothelial cell enrichment and RNA sequencing, we analyzed the gene expression of endothelial cells in mice, comparing brain endothelial cells to peripheral endothelial cells. We also assessed the regulation of CNS endothelial gene expression in models of stroke, multiple sclerosis, traumatic brain injury and seizures, each having profound BBB disruption. We found that although each is caused by a distinct trigger, they exhibit strikingly similar endothelial gene expression changes during BBB disruption, comprising a core BBB-dysfunction module that shifts the CNS endothelial cells into a peripheral endothelial cell-like state. The identification of a common pathway for BBB dysfunction suggests that targeting therapeutic agents to limit it may be effective across multiple neurological disorders.

Fluoride ingestion has been linked to changes in behavior in mice and rats, related to dose, sex of the animal, and the timing of exposure. Previous studies have shown the behavior of female rats to be most affected by postnatal fluoride exposure, and in this study we determined the effects of postnatal fluoride exposure on anxiety related behavior and serotonin. Mice given 50 ppm fluoride in drinking water had increased entries in the open arms of the elevated plus maze, suggesting reduced anxiety. Both peripheral and central serotonin was increased in the fluoride treated mice. In a cohort of children drinking water containing 2.5 ppm fluoride, serum serotonin was also increased as compared to controls. The mechanisms by which fluoride results in an increase peripheral and central serotonin are not well understood, but warrant further study, as these effects may also be relevant to prenatal fluoride related changes in behavior in both mice and humans.

Neurotrauma, a term referencing both traumatic brain and spinal cord injuries, is unique to neurodegeneration in that onset is clearly defined. From the perspective of matrix metalloproteinases (MMPs), there is opportunity to define their temporal participation in injury and recovery beginning at the level of the synapse. Here we examine the diverse roles of MMPs in the context of targeted insults (optic nerve lesion and hippocampal and olfactory bulb deafferentation), and clinically relevant focal models of traumatic brain and spinal cord injuries. Time-specific MMP postinjury signaling is critical to synaptic recovery after focal axonal injuries; members of the MMP family exhibit a signature temporal profile corresponding to axonal degeneration and regrowth, where they direct postinjury reorganization and synaptic stabilization. In both traumatic brain and spinal cord injuries, MMPs mediate early secondary pathogenesis including disruption of the blood-brain barrier, creating an environment that may be hostile to recovery. They are also critical players in wound healing including angiogenesis and the formation of an inhibitory glial scar. Experimental strategies to reduce their activity in the acute phase result in long-term neurological recovery after neurotrauma and have led to the first clinical trial in spinal cord injured pet dogs.

Background Cell based therapies, such as bone marrow derived mesenchymal stem cells (BM-MSCs; also known as mesenchymal stromal cells), are currently under investigation for a number of disease applications. The current challenge facing the field is maintaining the consistency and quality of cells especially for cell dose production for pre-clinical testing and clinical trials. Here we determine how BM-donor variability and thus the derived MSCs factor into selection of the optimal primary cell lineage for cell production and testing in a pre-clinical swine model of trauma induced acute respiratory distress syndrome. Methods We harvested bone marrow and generated three different primary BM-MSCs from Yorkshire swine. Cells from these three donors were characterized based on (a) phenotype (morphology, differentiation capacity and flow cytometry), (b) in vitro growth kinetics and metabolic activity, and (c) functional analysis based on inhibition of lung endothelial cell permeability. Results Cells from each swine donor exhibited varied morphology, growth rate, and doubling times. All expressed the same magnitude of standard MSC cell surface markers by flow cytometry and had similar differentiation potential. Metabolic activity and growth potential at each of the passages varied between the three primary cell cultures. More importantly, the functional potency of the MSCs on inhibition of endothelial permeability was also cell donor dependent. Conclusion This study suggests that for production of MSCs for cell-based therapy, it is imperative to examine donor variability and characterize derived MSCs for marker expression, growth and differentiation characteristics and testing potency in application dependent assays prior to selection of the optimal cell lineage for large scale expansion and dose production. Electronic supplementary material The online version of this article (10.1186/s12967-019-1877-4) contains supplementary material, which is available to authorized users.

BACKGROUND Plasma has been shown to mitigate the endotheliopathy of trauma. Protection of the endothelium may be due in part to fibrinogen and other plasma-derived proteins found in cryoprecipitate; however, the exact mechanisms remain unknown. Clinical trials are underway investigating early cryoprecipitate administration in trauma. In this study, we hypothesize that cryoprecipitate will inhibit endothelial cell (EC) permeability in vitro and will replicate the ability of plasma to attenuate pulmonary vascular permeability and inflammation induced by hemorrhagic shock and trauma (HS/T) in mice. METHODS In vitro, barrier permeability of ECs subjected to thrombin challenge was measured by transendothelial electrical resistance. In vivo, using an established mouse model of HS/T, we compared pulmonary vascular permeability among mice resuscitated with (1) lactated Ringer's solution (LR), (2) fresh frozen plasma (FFP), or (3) cryoprecipitate. Lung tissue from the mice in all groups was analyzed for markers of vascular integrity, inflammation, and inflammatory gene expression via NanoString messenger RNA quantification. RESULTS Cryoprecipitate attenuates EC permeability and EC junctional compromise induced by thrombin in vitro in a dose-dependent fashion. In vivo, resuscitation of HS/T mice with either FFP or cryoprecipitate attenuates pulmonary vascular permeability (sham, 297 ± 155; LR, 848 ± 331; FFP, 379 ± 275; cryoprecipitate, 405 ± 207; p < 0.01, sham vs. LR; p < 0.01, LR vs. FFP; and p < 0.05, LR vs. cryoprecipitate). Lungs from cryoprecipitate- and FFP-treated mice demonstrate decreased lung injury, decreased infiltration of neutrophils and activation of macrophages, and preserved pericyte-endothelial interaction compared with LR-treated mice. Gene analysis of lung tissue from cryoprecipitate- and FFP-treated mice demonstrates decreased inflammatory gene expression, in particular, IL-1β and NLRP3, compared with LR-treated mice. CONCLUSION Our data suggest that cryoprecipitate attenuates the endotheliopathy of trauma in HS/T similar to FFP. Further investigation is warranted on active components and their mechanisms of action.

While traumatic brain injury (TBI) is the leading cause of death and disability in children, we have yet to identify those pathogenic events that determine the extent of recovery. Neutrophils are best known as “first responders” to sites of infection and trauma where they become fully activated, killing pathogens via proteases that are released during degranulation. However, this activational state may generate substantial toxicity in the young brain after TBI that is partially due to developmentally regulated inadequate antioxidant reserves. Neutrophil degranulation is triggered via a downstream signaling pathway that is dependent on spleen tyrosine kinase (Syk). To test the hypothesis that the activational state of neutrophils is a determinant of early pathogenesis and long-term recovery, we compared young, brain-injured conditional knockouts of Syk (sykf/fMRP8-cre+) to congenic littermates (sykf/f). Based upon flow cytometry, there was an extended recruitment of distinct leukocyte subsets, including Ly6G+/Ly6C− and Ly6G+/Ly6Cint, over the first several weeks post-injury which was similar between genotypes. Subsequent assessment of the acutely injured brain revealed a reduction in blood-brain barrier disruption to both high and low molecular weight dextrans and reactive oxygen species in sykf/fMRP8-cre+ mice compared to congenic littermates, and this was associated with greater preservation of claudin 5 and neuronal integrity, as determined by Western blot analyses. At adulthood, motor learning was less affected in brain-injured sykf/fMRP8-cre+ mice as compared to sykf/f mice. Performance in the Morris Water Maze revealed a robust improvement in hippocampal-dependent acquisition and short and long-term spatial memory retention in sykf/fMRP8-cre+ mice. Subsequent analyses of swim path lengths during hidden platform training and probe trials showed greater thigmotaxis in brain-injured sykf/f mice than sham sykf/f mice and injured sykf/fMRP8-cre+ mice. Our results establish the first mechanistic link between the activation state of neutrophils and long-term functional recovery after traumatic injury to the developing brain. These results also highlight Syk kinase as a novel therapeutic target that could be further developed for the brain-injured child.

Background Hemorrhagic shock (HS) and trauma induce endothelial barrier compromise, inflammation, and aberrant clotting. We have shown that fresh human platelets (Plts) and Plt extracellular vesicles mitigate vascular leak in murine models of injury. Here, we investigate the potential of freeze-dried platelets (FDPlts) to attenuate pulmonary vascular permeability, decrease inflammation, and promote clotting in a murine model of HS. Methods Human FDPlts were characterized using in vitro assays of Plt marker expression, aggregation, coagulation, and endothelial cell permeability. An intravital model of vascular injury in the mouse cremaster muscle was used to assess the ability of FDPlts to incorporate into clots. Mouse groups subjected to controlled hemorrhage for 90 minutes were (1) lactated Ringer solution (LR), (2) FDPlts, (3) fresh human Plts, (4) murine whole blood (WB), and (5) shams (only instrumented). Hemorrhagic shock mouse endpoints included coagulation, pulmonary vascular permeability, and lung injury. Results Freeze-dried Plts expressed Plt-specific markers and retained functionality similar to fresh Plts. In in vitro assays of Plt aggregation, differences were noted. In vivo, FDPlts and Plts were found to incorporate into clots in postcapillary venules in the mouse cremaster muscle. Hemorrhagic shock mice resuscitated with LR displayed increased pulmonary vascular permeability compared with sham (sham, 686.6 ± 359.7; shock-LR, 2,637 ± 954.7; p = 0.001), and treatment with FDPlts or WB attenuated permeability compared with shock: shock-FDPlts, 1,328 ± 462.6 (p = 0.05), and shock-WB, 1,024 ± 370.5 (p = 0.0108). However, human Plts (Days 1-3) did not attenuate vascular leak in HS mice compared with shock-LR (shock-Plts, 3,601 ± 1,581; p = 0.33). Conclusion FDPlts contribute to clot formation similar to fresh human Plts. FDPlts also attenuated vascular permeability in vitro and in vivo. Mouse WB resuscitation but not fresh human Plts attenuated vascular permeability after HS. These data suggest that the effect of FDPlts may be a suitable alternative to fresh Plts in modulating hemostasis and the endotheliopathy associated with injury.

Background Mesenchymal stem cells (MSCs) have been shown to mitigate vascular permeability in hemorrhagic shock (HS) and trauma-induced brain and lung injury. Mechanistically, paracrine factors secreted from MSCs have been identified that can recapitulate many of the potent biologic effects of MSCs in animal models of disease. Interestingly, MSC-derived extracellular vesicles (EVs), contain many of these key soluble factors, and have therapeutic potential independent of the parent cells. In this study we sought to determine whether MSC-derived EVs (MSC EVs) could recapitulate the beneficial therapeutic effects of MSCs on lung vascular permeability induced by HS in mice. Methods Mesenchymal stem cell EVs were isolated from human bone marrow-derived MSCs by ultracentrifugation. A mouse model of fixed pressure HS was used to study the effects of shock, shock + MSCs and shock + MSC EVs on lung vascular endothelial permeability. Mice were administered MSCs, MSC EVs, or saline IV. Lung tissue was harvested and assayed for permeability, RhoA/Rac1 activation, and for differential phosphoprotein expression. In vitro, human lung microvascular cells junctional integrity was evaluated by immunocytochemistry and endothelial cell impedance assays. Results Hemorrhagic shock-induced lung vascular permeability was significantly decreased by both MSC and MSC EV infusion. Phosphoprotein profiling of lung tissue revealed differential activation of proteins and pathways related to cytoskeletal rearrangement and regulation of vascular permeability by MSCs and MSC EVs. Lung tissue from treatment groups demonstrated decreased activation of the cytoskeletal GTPase RhoA. In vitro, human lung microvascular cells, MSC CM but not MSC-EVs prevented thrombin-induced endothelial cell permeability as measured by electrical cell-substrate impedance sensing system and immunocytochemistry of VE-cadherin and actin. Conclusion Mesenchymal stem cells and MSC EVs modulate cytoskeletal signaling and attenuate lung vascular permeability after HS. Mesenchymal stem cell EVs may potentially be used as a novel "stem cell free" therapeutic to treat HS-induced lung injury.

Spinal cord injury (SCI) is often accompanied by reduced bladder compliance, which contributes to adverse conditions including urinary tract infections and vesicoureteral reflux. Reduced compliance is, in part, attributed to extensive remodeling of the bladder wall, including the extracellular matrix (ECM). Here, we tested the hypothesis that blockade of matrix metalloproteinases (MMPs), known for their ability to remodel the ECM, improves bladder compliance in dogs with SCI. We first evaluated dogs with naturally occurring SCIs resulting from intervertebral disc herniation (IVDH). After characterizing the natural history of urological recovery by cystometry in healthy dogs (n = 10) and dogs with SCIs (n = 20), we conducted a randomized, double-blinded, placebo-controlled clinical trial in dogs with IVDH-associated SCIs to assess the efficacy of the broad-spectrum MMP inhibitor, GM6001, given within 48 h post-injury. The primary outcomes were bladder compliance, as measured by cystometry, and an ordinal gait score (Texas Spinal Cord Injury Score; TSCIS) at day 42 post-SCI. Dogs (n = 93) were randomized to receive either dimethyl sulfoxide (DMSO) or GM6001+DMSO. There were transient, but significantly (p = 0.023) greater, adverse events (31 of 42; 74%) in the GM6001-treated group relative to vehicle controls (22 of 46; 48%). Whereas there were no differences in TSCIS between treatment groups at day 42 (p = 0.9679), bladder compliance was significantly higher in dogs treated with GM6001+DMSO compared to controls (p = 0.0272). Further studies are needed to determine whether this inhibition results from a direct interaction with the bladder wall or indirectly through neural-based mechanisms.

BACKGROUND:Platelet (Plt) derived-extracellular vesicles (Plt-EVs) have hemostatic properties similar to Plts. In addition to hemostasis, Plts also function to stabilize the vasculature and maintain endothelial cell (EC) barrier integrity. We hypothesized that Plt-EVs would inhibit vascular endothelial cell permeability, similar to fresh Plts. To investigate this hypothesis we utilized in vitro and in vivo models of vascular endothelial compromise and bleeding. METHODS:In vitro: Plt-EVs were isolated by ultracentrifugation and characterized for Plt markers and particle size distribution. Effects of Plts and Plt-EVs on endothelial barrier function was assessed by trans - endothelial electrical resistance (TEER) measurements and histological analysis of endothelial junction proteins. Hemostatic potential of Plt-EVs and Plts were assessed by multiple electrode Plt aggregometry. In vivo: The effects of Plts and Plt-EVs on vascular permeability and bleeding were assessed in NOD-SCID mice by an established Miles Assay of vascular permeability and a tail snip bleeding assay. RESULTS:In vitro: Plt-EVs displayed exosomal size distribution and expressed Plt specific surface markers. Plts and Plt-EVs decreased EC permeability and restored EC junctions after thrombin challenge. Multiplate aggregometry revealed that Plt-EVs enhanced Thrombin Receptor Activating Peptide (TRAP) mediated aggregation of whole blood, whereas Plts enhanced TRAP, Arachidonic Acid (ASPI), Collagen, and Adenosine Diphosphate (ADP) mediated aggregation. In vivo: Plt-EVs are equivalent to Plts in attenuating VEGF-A induced vascular permeability and uncontrolled blood loss in a tail snip hemorrhage model. CONCLUSION:Our study is the first to report that Plt-EVs might provide a feasible product for transfusion in trauma patients to attenuate bleeding, inhibit vascular permeability and mitigate the endotheliopathy of trauma (EOT). STUDY TYPE:Original Article LEVEL OF EVIDENCE: This is a pre-clinical study so it does not confirm to the level of evidence table for all clinical studies and case reports.

Neuropathic pain and bladder dysfunction represent significant quality-of-life issues for many spinal cord injury patients. Loss of GABAergic tone in the injured spinal cord may contribute to the emergence of these symptoms. Previous studies have shown that transplantation of rodent inhibitory interneuron precursors from the medial ganglionic eminence (MGE) enhances GABAergic signaling in the brain and spinal cord. Here we look at whether transplanted MGE-like cells derived from human embryonic stem cells (hESC-MGEs) can mitigate the pathological effects of spinal cord injury. We find that 6 months after transplantation into injured mouse spinal cords, hESC-MGEs differentiate into GABAergic neuron subtypes and receive synaptic inputs, suggesting functional integration into host spinal cord. Moreover, the transplanted animals show improved bladder function and mitigation of pain-related symptoms. Our results therefore suggest that this approach may be a valuable strategy for ameliorating the adverse effects of spinal cord injury.

Matrix metalloproteinase (MMP)-2 knockout (KO) mice show impaired neurological recovery after spinal cord injury (SCI), suggesting that this proteinase is critical to recovery processes. However, this finding in the KO has been confounded by a compensatory increase in MMP-9. We synthesized the thiirane mechanism-based inhibitor ND-378 and document that it is a potent (nanomolar) and selective slow-binding inhibitor of MMP-2 that does not inhibit the closely related MMP-9 and MMP-14. ND-378 crosses the blood-spinal cord barrier, achieving therapeutic concentrations in the injured spinal cord. Spinal-cord injured mice treated with ND-378 showed no change in long-term neurological outcomes, suggesting that MMP-2 is not a key determinant of locomotor recovery.

While neutrophil elastase (NE), released by activated neutrophils, is a key mediator of secondary pathogenesis in adult models of brain ischemia and spinal cord injury, no studies to date have examined this protease in the context of the injured immature brain, where there is notable vulnerability resulting from inadequate antioxidant reserves and prolonged exposure to infiltrating neutrophils. We thus reasoned that NE may be a key determinant of secondary pathogenesis, and as such, adversely influence long-term neurological recovery. To address this hypothesis, wild-type (WT) and NE knockout (KO) mice were subjected to a controlled cortical impact at post-natal day 21, approximating a toddler-aged child. To determine if NE is required for neutrophil infiltration into the injured brain, and whether this protease contributes to vasogenic edema, we quantified neutrophil numbers and measured water content in the brains of each of these genotypes. While leukocyte trafficking was indistinguishable between genotypes, vasogenic edema was markedly attenuated in the NE KO. To determine if early pathogenesis is dependent on NE, indices of cell death (TUNEL and activated caspase-3) were quantified across genotypes. NE KO mice showed a reduction in these markers of cell death in the injured hippocampus, which corresponded to greater preservation of neuronal integrity as well as reduced expression of heme oxygenase-1, a marker of oxidative stress. WT mice, treated with a competitive inhibitor of NE at 2, 6 and 12 h post-injury, likewise showed a reduction in cell death and oxidative stress compared to vehicle-treated controls. We next examined the long-term behavioral and structural consequences of NE deficiency. NE KO mice showed an improvement in long-term spatial memory retention and amelioration of injury-induced hyperactivity. However, volumetric and stereological analyses found comparable tissue loss in the injured cortex and hippocampus independent of genotype. Further, WT mice treated acutely with the NE inhibitor showed no long-term behavioral or structural improvements. Together, these findings validate the central role of NE in both acute pathogenesis and chronic functional recovery, and support future exploration of the therapeutic window, taking into account the prolonged period of neutrophil trafficking into the injured immature brain.

Angiogenesis plays a critical role in wound healing after spinal cord injury. Therefore, understanding the events that regulate angiogenesis has considerable relevance from a therapeutic standpoint. We evaluated the contribution of matrix metalloproteinase (MMP)-2 to angiogenesis and vascular stability in spinal cord injured MMP-2 knockout and wildtype (WT) littermates. While MMP-2 deficiency resulted in reduced endothelial cell division within the lesioned epicenter, there were no genotypic differences in vascularity (vascular density, vascular area, and endothelial cell number) over the first two weeks post-injury. However, by 21days post-injury MMP-2 deficiency resulted in a sharp decline in vascularity, indicative of vascular regression. Complementary in vitro studies of brain capillary endothelial cells confirmed MMP-2 dependent proliferation and tube formation. As deficiency in MMP-2 led to prolonged MMP-9 expression in the injured spinal cord, we examined both short-term and long-term exposure to MMP-9 in vitro. While MMP-9 supported endothelial tube formation and proliferation, prolonged exposure resulted in loss of tubes, findings consistent with vascular regression. Vascular instability is frequently associated with pericyte dissociation and precedes vascular regression. Quantification of PDGFrβ+ pericyte coverage of mature vessels within the glial scar (the reactive gliosis zone), a known source of MMP-9, revealed reduced coverage in MMP-2 deficient animals. These findings suggest that acting in the absence of MMP-2, MMP-9 transiently supports angiogenesis during the early phase of wound healing while its prolonged expression leads to vascular instability and regression. These findings should be considered while developing therapeutic interventions that block MMPs.

The infiltration of monocytes into the lesioned site is a key event in the inflammatory response after spinal cord injury (SCI). We hypothesized that the molecular events governing the infiltration of monocytes into the injured cord involve cooperativity between the upregulation of the chemoattractant stromal cell-derived factor-1 (SDF-1)/CXCL12 in the injured cord and matrix metalloproteinase-9 (MMP-9/gelatinase B), expressed by infiltrating monocytes. SDF-1 and its receptor CXCR4 mRNAs were upregulated in the injured cord, while macrophages immunoexpressed CXCR4. When mice, transplanted with bone marrow cells from green fluorescent protein (GFP) transgenic mice, were subjected to SCI, GFP+ monocytes infiltrated the cord and displayed gelatinolytic activity.In vitrostudies confirmed that SDF-1α, acting through CXCR4, expressed on bone marrow-derived macrophages, upregulated MMP-9 and stimulated MMP-9-dependent transmigration across endothelial cell monolayers by 2.6-fold. There was a reduction in F4/80+ macrophages in spinal cord-injured MMP-9 knock-out mice (by 36%) or wild-type mice, treated with the broad-spectrum MMP inhibitor GM6001 (by 30%). Mice were adoptively transferred with myeloid cells and treated with the MMP-9/-2 inhibitor SB-3CT, the CXCR4 antagonist AMD3100, or a combination of both drugs. While either drug resulted in a 28–30% reduction of infiltrated myeloid cells, the combined treatment resulted in a 45% reduction, suggesting that SDF-1 and MMP-9 function independently to promote the trafficking of myeloid cells into the injured cord. Collectively, these observations suggest a synergistic partnership between MMP-9 and SDF-1 in facilitating transmigration of monocytes into the injured spinal cord.

L-selectin, a lectin-like receptor on all leukocyte classes, functions in adhesive and signaling roles in the recruitment of myeloid cells from the blood to sites of inflammation. Here, we consider L-selectin as a determinant of neurological recovery in a murine model of spinal cord injury (SCI). Spinal cord-injured, L-selectin knock-out (KO) mice (male) showed improved long-term recovery with greater white matter sparing relative to wild-type (WT) mice and reduced oxidative stress in the injured cord at 72 h post-SCI. There was a partial and transient reduction in accumulation of neutrophils in the injured spinal cords of KOs at 24 h post-injury. To complement these findings with KO mice, we sought a pharmacologic means for lowering L-selectin levels. We found that diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), induced the shedding of L-selectin from the cell surface of myeloid subsets, specifically neutrophils and non-classical monocytes, in the blood and the injured spinal cord. Diclofenac administration to injured WT mice enhanced neurological recovery to a level comparable to that of KOs but did not improve recovery in KOs. While diclofenac treatment had no effect on myeloid cell accumulation, there was a reduction in oxidative stress at 72 h post-SCI. These findings implicate L-selectin in secondary pathogenesis beyond a role in leukocyte recruitment and raise the possibility of repurposing diclofenac for the treatment of SCI.

Objective Mice subjected to traumatic brain injury at postnatal day 21 show emerging cognitive deficits that coincide with hippocampal neuronal loss. Here we consider glutathione peroxidase (GPx) activity as a determinant of recovery in the injured immature brain. Methods Wild-type and transgenic (GPxTg) mice overexpressing GPx were subjected to traumatic brain injury or sham surgery at postnatal day 21. Animals were killed acutely (3 or 24 hours after injury) to assess oxidative stress and cell injury in the hippocampus or 4 months after injury after behavioral assessments. Results In the acutely injured brains, a reduction in oxidative stress markers including nitrotyrosine was seen in the injured GPxTg group relative to wild-type control mice. In contrast, cell injury, with marked vulnerability in the dentate gyrus, was apparent despite no differences between genotypes. Magnetic resonance imaging demonstrated an emerging cortical lesion during brain maturation that was also indistinguishable between injured genotypes. Stereological analyses of cortical volumes likewise confirmed no genotypic differences between injured groups. However, behavioral tests beginning 3 months after injury demonstrated improved spatial memory learning in the GPxTg group. Moreover, stereological analysis within hippocampal subregions demonstrated a significantly greater number of neurons within the dentate of the GPx group. Interpretation Our results implicate GPx in recovery of spatial memory after traumatic brain injury. This recovery may be attributed, in part, to a reduction in early oxidative stress and selective, long-term sparing of neurons in the dentate. Ann Neurol 2009;65:540–549

The immune response that accompanies spinal cord injury contributes to both injury and reparative processes. It is this duality that is the focus of this review. Here we consider the complex cellular and molecular immune responses that lead to the infiltration of leukocytes and glial activation, promote oxidative stress and tissue damage, influence wound healing, and subsequently modulate locomotor recovery. Immunomodulatory strategies to improve outcomes are gaining momentum as ongoing research carefully dissects those pathways, which likely mediate cell injury from those, which favor recovery processes. Current therapeutic strategies address divergent approaches including early immunoblockade and vaccination with immune cells to prevent early tissue damage and support a wound-healing environment that favors plasticity. Despite these advances, there remain basic questions regarding how inflammatory cells interact in the injured spinal cord. Such questions likely arise as a result of our limited understanding of immune cell/neural interactions in a dynamic environment that culminates in progressive cell injury, demyelination, and regenerative failure.

Cell-based gene delivery for gene therapy offers the advantages of long-term stable expression of proteins without the safety concerns associated with viral vectors. However, issues of immune rejection prevent the widespread use of allogeneic cell implants. In this study, we determine if Sertoli cells, known for their immune privileged status, are suitable vehicles for allogeneic cell-based gene delivery into the injured spinal cord. As proof of concept, Sertoli cells were modified with recombinant adenovirus expressing enhanced green fluorescent protein (eGFP) or a human trophic factor, neurotrophin-3 (hNT-3), and eGFP. Genetically modified Sertoli cells retained their immunosuppressive ability in vitro, based upon lymphocyte proliferation assays, and were capable of generating biologically relevant levels of NT-3. Similarly, modified, allogeneic cells, implanted into the acutely injured spinal cord, reduced the early inflammatory response while producing significant levels of hNT-3 for at least 3 days after grafting. Moreover, these cells survived for at least 42 days after implantation in the injured cord. Together, these results demonstrate that Sertoli cells function in immunomodulation, can be engineered to produce bioactive molecules, and show long-term survival after implantation into the hostile environment of the acutely injured spinal cord. Such long-term survival represents an important first step toward developing an optimal cell-based delivery system that generates sustained expression of a therapeutic molecule.

Matrix metalloproteinase-9 is elevated within the acutely injured murine spinal cord and blockade of this early proteolytic activity with GM6001, a broad-spectrum matrix metalloproteinase inhibitor, results in improved recovery after spinal cord injury. As matrix metalloproteinase-9 is likewise acutely elevated in dogs with naturally occurring spinal cord injuries, we evaluated efficacy of GM6001 solubilized in dimethyl sulfoxide in this second species. Safety and pharmacokinetic studies were conducted in naïve dogs. After confirming safety, subsequent pharmacokinetic analyses demonstrated that a 100 mg/kg subcutaneous dose of GM6001 resulted in plasma concentrations that peaked shortly after administration and were sustained for at least 4 days at levels that produced robust in vitro inhibition of matrix metalloproteinase-9. A randomized, blinded, placebo-controlled study was then conducted to assess efficacy of GM6001 given within 48 hours of spinal cord injury. Dogs were enrolled in 3 groups: GM6001 dissolved in dimethyl sulfoxide (n = 35), dimethyl sulfoxide (n = 37), or saline (n = 41). Matrix metalloproteinase activity was increased in the serum of injured dogs and GM6001 reduced this serum protease activity compared to the other two groups. To assess recovery, dogs were a priori stratified into a severely injured group and a mild-to-moderate injured group, using a Modified Frankel Scale. The Texas Spinal Cord Injury Score was then used to assess long-term motor/sensory function. In dogs with severe spinal cord injuries, those treated with saline had a mean motor score of 2 (95% CI 0-4.0) that was significantly (P

While a number of studies have examined the complex roles of leukocytes in the acute and chronically injured cord, few have specifically focused on neutrophils, where we have only recently begun to appreciate their involvement in both vascular pathogenesis and early wound healing. Here we address the mechanisms underlying neutrophil-mediated endothelial destabilization, their synergism with monocytes in modulating permeability, and their putative role as initiators of angiogenesis in the acutely injured spinal cord. Neutrophils contain a variety of bioactive molecules that are stored in granules. Studies have shown that certain of these molecules, and most notably proteases, contribute to endothelial destabilization as neutrophils degranulate during their transmigration across this front. Neutrophils have historically been regarded as detrimental to the acutely injured cord. However, there is growing evidence that this may be an oversimplified view as it fails to take into account their ability to release proteases that degrade the extracellular matrix, releasing latent growth factors that may in turn support early angiogenesis.

We have investigated the role of the TATA-binding protein (TBP) in modulating RNA polymerase (Pol) III gene activity. Epitope-tagged TBP (e-TBP) was both transiently and stably transfected inDrosophilaSchneider S-2 cells to increase the total cellular level of TBP. Analysis of the transcripts synthesized from cotransfected tRNA and U6 RNA genes revealed that both types of RNA Pol III promoters were substantially stimulated by an increase in e-TBP in a dose-dependent manner. Furthermore, a TBP-dependent increase in the levels of endogenous tRNA transcripts was produced in the stable line induced to express the e-TBP. We further determined whether the ability of increased TBP to induce RNA Pol III gene expression was due to a direct effect of increased TBP complexes on RNA Pol III gene promoters or an indirect consequence of enhanced expression of RNA Pol II genes. A TBP expression plasmid (e-TBP332), containing a mutation within the highlyconservedcarboxy-terminaldomain,wasbothtransientlyandstablytransfectedintoS-2cells.e-TBP332 augmented the transcription from two RNA Pol II gene promoters indistinguishably from that observed when e-TBPwasexpressed.Incontrast,e-TBP332wascompletelydefectiveinitsabilitytostimulateeitherthetRNA or U6 RNA gene promoters. In addition, increasing levels of a truncated TBP protein containing only the carboxy-terminal region failed to induce either the tRNA or U6 RNA gene promoter, whereas it retained its ability to stimulate an RNA Pol II promoter. Thus, the TBP-dependent increase in RNA Pol II gene activity is not sufficient for enhanced RNA Pol III gene transcription; rather, a direct effect on RNA Pol III promoters is required. Furthermore, these results provide thefirst direct evidence that the amino-terminal region of TBP is important for the formation or function of TBP-containing complexes utilized by TATA-less and TATAcontainingRNAPolIIIpromoters.Together,thesestudiesdemonstratethatTBPislimitingfortheexpression of both classes of RNA Pol III promoters in Drosophila cells and implicate an important role for TBP in regulating RNA Pol III gene expression. RNA polymerase (Pol) III is responsible for the transcription of many small cellular and viral RNAs which are untranslated. There are three major classes of promoters that dictate the expression of the genes transcribed by RNA Pol III (43). The tRNA and 5S RNA classes of promoters each contain intragenic promoter elements and generally lack TATA sequences upstream of the gene. Both of these types of promoters require TFIIIB and TFIIIC components to reconstitute transcription in vitro, while the 5S RNA gene additionally uses

It has long been thought that mammalian Sertoli cells are terminally differentiated and nondividing postpuberty. For most previous in vitro studies immature rodent testes have been the source of Sertoli cells and these have shown little proliferative ability when cultured. We have isolated and characterized Sertoli cells from human cadaveric testes from seven donors ranging from 12 to 36 years of age. The cells proliferated readily in vitro under the optimized conditions used with a doubling time of approximately 4 days. Nuclear 5-ethynyl-2´-deoxyuridine (EdU) incorporation confirmed that dividing cells represented the majority of the population. Classical Sertoli cell ultrastructural features, lipid droplet accumulation, and immunoexpression of GATA-4, Sox9, and the FSH receptor (FSHr) were observed by electron and fluorescence microscopy, respectively. Flow cytometry revealed the expression of GATA-4 and Sox9 by more than 99% of the cells, and abundant expression of a number of markers indicative of multipotent mesenchymal cells. Low detection of endogenous alkaline phosphatase activity after passaging showed that few peritubular myoid cells were present. GATA-4 and SOX9 expression were confirmed by reverse transcription polymerase chain reaction (RT-PCR), along with expression of stem cell factor (SCF), glial cell line-derived neurotrophic factor (GDNF), and bone morphogenic protein 4 (BMP4). Tight junctions were formed by Sertoli cells plated on transwell inserts coated with fibronectin as revealed by increased transepithelial electrical resistance (TER) and polarized secretion of the immunoregulatory protein, galectin-1. These primary Sertoli cell populations could be expanded dramatically in vitro and could be cryopreserved. The results show that functional human Sertoli cells can be propagated in vitro from testicular cells isolated from adult testis. The proliferative human Sertoli cells should have important applications in studying infertility, reproductive toxicology, testicular cancer, and spermatogenesis, and due to their unique biological properties potentially could be useful in cell therapy. Fil: Chui, Kitty. MandalMed Inc. ; Estados Unidos Fil: Trivedi, Alpa. MandalMed Inc.; Estados Unidos. University of California; Estados Unidos Fil: Cheng, C. Yan. Lonza Walkersville; Estados Unidos Fil: Cherbavaz, Diana B. . MandalMed Inc.; Estados Unidos Fil: Dazin, Paul F.. MandalMed; Estados Unidos Fil: Huynh, Ai Lam Thu. MandalMed Inc.; Estados Unidos Fil: Mitchel, James B.. Lonza Walkersville; Estados Unidos Fil: Rabinovich, Gabriel Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina Fil: Noble Haeusslein, Linda J.. University of California; Estados Unidos Fil: John, Constance M.. MandalMed Inc.; Estados Unidos

Injuries to the central and peripheral nervous system result in varying degrees of paresis and as such alter gait. We developed novel quantitative measures to assess compensatory patterns of gait in experimental models of unilateral and bilateral hindlimb paresis. We hypothesized that hindlimb paresis results in unique alterations in the gait cycle that reflect the symmetry of the initial lesion. To test this hypothesis, adult, male Sprague-Dawley rats were subjected to a unilateral sciatic nerve crush injury or a moderately severe spinal cord contusion injury at T8. Kinematic and timing parameters were captured simultaneously in all four limbs and alterations in gait were then compared to relevant sham controls. Gait analysis consisted of walking trials through a gait tunnel positioned over a Tekscan pressure sensor grid. After sciatic nerve injury, animals unweighted the injured limb by shifting their center of mass toward the contralateral forelimb and hindlimb. These changes in weight-bearing occurred simultaneously with an increase in stance time on the contralateral limbs. As might be expected spinal cord injured animals unweighted their hindlimbs, as shown by reduced hindlimb contact force and contact pressure. These adjustments coincided with a shortening of forelimb stance time and stride length. These findings show both alterations and compensatory changes in gait that reflect the symmetry of the initial injury.

The gelatinases, matrix metalloproteinases (MMP)-2 and MMP-9, are thought to be key mediators of secondary damage in adult animal models of brain injury. Moreover, an acute increase in these proteases in plasma and brain extracellular fluid of adult patients with moderate-to-severe traumatic brain injuries (TBIs) is associated with poorer clinical outcomes and mortality. Nonetheless, their involvement after TBI in the pediatric brain remains understudied. Using a murine model of TBI at postnatal day 21 (p21), approximating a toddler-aged child, we saw upregulation of active and pro-MMP-9 and MMP-2 by gelatin zymography at 48 h post-injury. We therefore investigated the role of gelatinases on long-term structural and behavioral outcomes after injury after acute inhibition with a selective gelatinase inhibitor, p-OH SB-3CT. After systemic administration, p-OH SB-3CT crossed the blood-brain barrier at therapeutically-relevant concentrations. TBI at p21 induced hyperactivity, deficits in spatial learning and memory, and reduced sociability when mice were assessed at adulthood, alongside pronounced tissue loss in key neuroanatomical regions. Acute and short-term post-injury treatment with p-OH SB-3CT did not ameliorate these long-term behavioral, cognitive, or neuropathological deficits as compared to vehicle-treated controls, suggesting that these deficits were independent of MMP-9 and MMP-2 upregulation. These findings emphasize the vulnerability of the immature brain to the consequences of traumatic injuries. However, early upregulation of gelatinases do not appear to be key determinants of long-term recovery after an early-life injury.

In contrast to yeast and mammalian systems, which depend principally on internal promoter elements for tRNA gene transcription, insect systems require additional upstream sequences. To understand the function of the upstream sequences, we have asked whether the Bombyx mori tRNACAla and tRNASGAla genes, which are absolutely dependent on these sequences in vitro, also require them for transcription in vivo. We introduced wild-type and mutant versions of the Bombyx tRNAAla genes into Drosophila Schneider-2 cells and found that the tRNACAla gene is efficiently transcribed and that its transcription depends strongly on the distal segment of its upstream promoter. In contrast, the tRNASGAla gene is inefficiently transcribed, and this inefficiency results from lack of a specific sequence within the distal tRNACAla upstream promoter. This sequence, 5'-TTTATAT-3', is sufficient to increase the activity of the tRNASGAla promoter to that of the tRNACAla promoter. Moreover, promoters containing the 5'-TTTATAT-3' element are stimulated by increased levels of cellular TATA-binding protein. Together these results indicate that, in insect cells, a TATA-like element is specifically required to form functional TATA-binding protein-containing complexes that promote efficient transcription of tRNA genes.

The hepatitis B virus (HBV) X protein is essential for viral infectivity, and evidence indicates that it is a strong contributor to HBV-mediated oncogenesis. X has been shown to transactivate a wide variety of RNA polymerase (Pol) II-dependent, as well as RNA Pol III-dependent, promoters. In this study, we have investigated the possibility that X modulates RNA Pol I-dependent rRNA transcription. In both human hepatoma Huh7 and Drosophila Schneider S2 cell lines, X expression stimulated rRNA promoter activity. Extracts prepared from X-expressing cells stably transfected with an X gene also exhibited an increased ability to transcribe the rRNA promoter. The mechanism for X transactivation was examined by determining whether this regulatory event was dependent on Ras activation and increased TATA-binding protein (TBP) levels. Our previous studies have demonstrated that X, and the activation of Ras, produces an increase in the cellular levels of TBP (H.-D. Wang, A. Trivedi, and D. L. Johnson, Mol. Cell. Biol. 17:6838-6846, 1997). Expression of a dominant negative form of Ras blocked the X-mediated induction of the rRNA promoters, whereas expression of a constitutively activated form of Ras mimicked the enhancing effect of X on rRNA promoter activity. When TBP was overexpressed in either Huh7 or S2 cells, a dose-dependent increase in rRNA promoter activity was observed. To analyze whether the increase in TBP was modulating rRNA promoter activity indirectly, by increasing activity of RNA Pol II-dependent promoters, a Drosophila TBP cDNA was constructed with a mutation that eliminated its ability to stimulate RNA Pol II-dependent promoters. Transient expression of wild-type TBP in S2 cells increased the activities of specific RNA Pol I- and Pol II-dependent promoters. Expression of the mutant TBP protein failed to enhance the activity of the RNA Pol II-dependent promoters, yet the protein completely retained its ability to stimulate the rRNA promoter. Furthermore, the addition of recombinant TBP to S2 extracts stimulated rRNA promoter activity in vitro. Together, these results demonstrate that the HBV X protein up-regulates RNA Pol I-dependent promoters via a Ras-activated pathway in two distinct cell lines. The enhanced promoter activity can, at least in part, be attributed to the X- and Ras-mediated increase in cellular TBP, a limiting transcription component.

Our previous studies have shown that the hepatitis B virus protein, X, activates all three classes of RNA polymerase III (pol III)-dependent promoters by increasing the cellular level of TATA-binding protein (TBP) (H.-D. Wang et al., Mol. Cell. Biol. 15:6720-6728, 1995), a limiting transcription component (A. Trivedi et al., Mol. Cell. Biol. 16:6909-6916, 1996). We have investigated whether these X-mediated events are dependent on the activation of the Ras/Raf-1 signaling pathway. Transient expression of a dominant-negative mutant Ras gene (Ras-ala15) in a Drosophila S-2 stable cell line expressing X (X-S2), or incubation of the cells with a Ras farnesylation inhibitor, specifically blocked both the X-dependent activation of a cotransfected tRNA gene and the increase in cellular TBP levels. Transient expression of a constitutively activated form of Ras (Ras-val12) in control S2 cells produced both an increase in tRNA gene transcription and an increase in cellular TBP levels. These events are not cell type specific since X-mediated gene induction was also shown to be dependent on Ras activation in a stable rat 1A cell line expressing X. Furthermore, increases in RNA pol III-dependent gene activity and TBP levels could be restored in X-S2 cells expressing Ras-ala15 by coexpressing a constitutively activated form of Raf-1. These events are serum dependent, and when the cells are serum deprived, the X-mediated effects are augmented. Together, these results demonstrate that the X-mediated induction of RNA pol III-dependent genes and increase in TBP are both dependent on the activation of the Ras/Raf-1 signaling cascade. In addition, these studies define two new and important consequences mediated by the activation of the Ras signal transduction pathway: an increase in the central transcription factor, TBP, and the induction of RNA pol III-dependent gene activity.