Factor XII inhibition reduces thrombus formation in a primate thrombosis model.

The plasma zymogens factor XII (fXII) and factor XI (fXI) contribute to thrombosis in a variety of mouse models. These proteins serve a limited role in hemostasis, suggesting that antithrombotic therapies targeting them may be associated with low bleeding risks. Although there is substantial epidemiologic evidence supporting a role for fXI in human thrombosis, the situation is not as clear for fXII. We generated monoclonal antibodies (9A2 and 15H8) against the human fXII heavy chain that interfere with fXII conversion to the protease factor XIIa (fXIIa). The anti-fXII antibodies were tested in models in which anti-fXI antibodies are known to have antithrombotic effects. Both anti-fXII antibodies reduced fibrin formation in human blood perfused through collagen-coated tubes. fXII-deficient mice are resistant to ferric chloride-induced arterial thrombosis, and this resistance can be reversed by infusion of human fXII. 9A2 partially blocks, and 15H8 completely blocks, the prothrombotic effect of fXII in this model. 15H8 prolonged the activated partial thromboplastin time of baboon and human plasmas. 15H8 reduced fibrin formation in collagen-coated vascular grafts inserted into arteriovenous shunts in baboons, and reduced fibrin and platelet accumulation downstream of the graft. These findings support a role for fXII in thrombus formation in primates.

Inhibition of factor XI activation attenuates inflammation and coagulopathy while improving the survival of mouse polymicrobial sepsis.

Severe bacterial sepsis often leads to a systemic procoagulant and proinflammatory condition that can manifest as disseminated intravascular coagulation, septic shock, and multiple organ failure. Because activation of the contact proteases factor XII (FXII), prekallikrein, and factor XI (FXI) can trigger coagulation and inflammatory responses, the contact factors have been considered potential targets for the treatment of sepsis. However, the pathogenic role of contact activation in severe infections has not been well defined. We therefore investigated whether an anticoagulant antibody (14E11) that selectively inhibits prothrombotic FXI activation by activated FXII (FXIIa) modifies the course of bowel perforation-induced peritoneal sepsis in mice. Early anticoagulation with 14E11 suppressed systemic thrombin- antithrombin complex formation, IL-6, and TNF-α levels, and reduced platelet consumption in the circulation and deposition in the blood vessels. Treatment with 14E11 within 12 hours after bowel perforation significantly improved survival compared with vehicle treatment, and the saturating dose did not increase tail bleeding. These data suggest that severe polymicrobial abdominal infection induces prothrombotic FXI activation, to the detriment of the host. Systemic anticoagulation by inhibiting FXI activation or FXIIa procoagulant activity during sepsis may therefore limit the development of disseminated intravascular coagulation without increasing bleeding risks.

Establishing the normal range of sperm DNA fragmentation index (% DFI) for rhesus macaques.

The Sperm Chromatin Structure Assay (SCSA) is a robust test with high repeatability and precision. It is a clinically accepted assay that defines risk for infertility in men by measuring the degree of DNA fragmentation (% DFI) in sperm. The objective of this study was to adapt and validate the SCSA for rhesus macaques (Macaca mulatta) and establish a range for % DFI in fertile males. Sperm samples from two different males were used to produce a % DFI validation curve before establishing a range using additional samples from n = 11 males. Sperm labeled with acridine orange were analyzed by flow cytometry to measure green fluorescence (native or intact DNA) and red fluorescence (fragmented DNA). Data were exported to FlowJo software to determine the % DFI for each sample. DNA fragmentation ranged from 0.1 to 2.4% DFI, with a mean ± SD = 1.1 ± 0.7% DFI (validation curve optimized to R2 > 0.95). In conclusion, we were able to successfully validate the SCSA in our institution and establish the first normal range for sperm DNA fragmentation in rhesus macaques. Our study provides a quantitative baseline for future evaluations to assess macaque fertility through the SCSA test.

Multiple preconditioning paradigms converge on interferon regulatory factor-dependent signaling to promote tolerance to ischemic brain injury.

Ischemic tolerance can be induced by numerous preconditioning stimuli, including various Toll-like receptor (TLR) ligands. We have shown previously that systemic administration of the TLR4 ligand LPS or the TLR9 ligand unmethylated CpG oligodeoxynucleotide before transient brain ischemia in mice confers substantial protection against ischemic damage. To elucidate the molecular mechanisms of preconditioning, we compared brain genomic profiles in response to preconditioning with these TLR ligands and with preconditioning via exposure to brief ischemia. We found that exposure to the TLR ligands and brief ischemia induced genomic changes in the brain characteristic of a TLR pathway-mediated response. Interestingly, all three preconditioning stimuli resulted in a reprogrammed response to stroke injury that converged on a shared subset of 13 genes not evident in the genomic profile from brains that were subjected to stroke without prior preconditioning. Analysis of the promoter region of these shared genes showed sequences required for interferon regulatory factor (IRF)-mediated transcription. The importance of this IRF gene network was tested using mice deficient in IRF3 or IRF7. Our data show that both transcription factors are required for TLR-mediated preconditioning and neuroprotection. These studies are the first to discover a convergent mechanism of neuroprotection induced by preconditioning--one that potentially results in reprogramming of the TLR-mediated response to stroke and requires the presence of IRF3 and IRF7.

Toll-like receptor 7 preconditioning induces robust neuroprotection against stroke by a novel type I interferon-mediated mechanism.

BACKGROUND AND PURPOSE: Systemic administration of Toll-like receptor (TLR) 4 and TLR9 agonists before cerebral ischemia have been shown to reduce ischemic injury by reprogramming the response of the brain to stroke. Our goal was to explore the mechanism of TLR-induced neuroprotection by determining whether a TLR7 agonist also protects against stroke injury. METHODS: C57Bl/6, TNF(-/-), interferon (IFN) regulatory factor 7(-/-), or type I IFN receptor (IFNAR)(-/-) mice were subcutaneously administered the TLR7 agonist Gardiquimod (GDQ) 72 hours before middle cerebral artery occlusion. Infarct volume and functional outcome were determined after reperfusion. Plasma cytokine responses and induction of mRNA for IFN-related genes in the brain were measured. IFNAR(-/-) mice also were treated with the TLR4 agonist (lipopolysaccharide) or the TLR9 agonist before middle cerebral artery occlusion and infarct volumes measured. RESULTS: The results show that GDQ reduces infarct volume as well as functional deficits in mice. GDQ pretreatment provided robust neuroprotection in TNF(-/-) mice, indicating that TNF was not essential. GDQ induced a significant increase in plasma IFNα levels and both IRF7(-/-) and IFNAR(-/-) mice failed to be protected, implicating a role for IFN signaling in TLR7-mediated protection. CONCLUSIONS: Our studies provide the first evidence that TLR7 preconditioning can mediate neuroprotection against ischemic injury. Moreover, we show that the mechanism of protection is unique from other TLR preconditioning ligands in that it is independent of TNF and dependent on IFNAR.

Increased expression of the close homolog of the adhesion molecule L1 in different cell types over time after rat spinal cord contusion.

The close homolog of the adhesion molecule L1 (CHL1) is important during CNS development, but a study with CHL1 knockout mice showed greater functional recovery after spinal cord injury (SCI) in its absence. We investigated CHL1 expression from 1 to 28 days after clinically relevant contusive SCI in Sprague-Dawley rats. Western blot analysis showed that CHL1 expression was significantly up-regulated at day 1 and further increased over 4 weeks after SCI. Immunohistochemistry of tissue sections showed that CHL1 in the intact spinal cord was expressed at low levels. By 1 day and through 4 weeks after SCI, CHL1 became highly expressed in NG2(+) cells. Hypertrophic GFAP(+) astrocytes also expressed CHL1 by 1 week after injury. The increase in CHL1 protein paralleled that of NG2 in the first week and GFAP between 1 and 4 weeks after injury. At 4 weeks, NG2(+) /CHL1(+) cells and GFAP(+) /CHL1(+) astrocytes were concentrated at the boundary between residual spinal cord tissue and the central lesion. NF200(+) spinal cord axons approached but did not penetrate this boundary. In contrast, CHL1(+) cells in the central lesion at 1 week and later colabeled with p75 and NG2 and were chronically associated with many NF200(+) axons, presumably axons that had sprouted in association with CHL1(+) Schwann cells infiltrating the cord after contusion. Thus, our study demonstrates up-regulation of CHL1 in multiple cell types and locations in a rat model of contusion injury and suggests that this molecule may be involved both in inhibition of axonal regeneration and in recovery processes after SCI.

Systemic lipopolysaccharide protects the brain from ischemic injury by reprogramming the response of the brain to stroke: a critical role for IRF3.

Lipopolysaccharide (LPS) preconditioning provides neuroprotection against subsequent cerebral ischemic injury through activation of its receptor, Toll-like receptor 4 (TLR4). Paradoxically, TLR activation by endogenous ligands after ischemia worsens stroke damage. Here, we define a novel, protective role for TLRs after ischemia in the context of LPS preconditioning. Microarray analysis of brains collected 24 h after stroke revealed a unique set of upregulated genes in LPS-pretreated animals. Promoter analysis of the unique gene set identified an overrepresentation of type I interferon (IFN)-associated transcriptional regulatory elements. This finding suggested the presence of type I IFNs or interferon regulatory factors (IRFs), which upregulate interferon-stimulated genes. Upregulation of IFNbeta was confirmed by real-time reverse transcription-PCR. Direct administration of IFNbeta intracerebroventricularly at the time of stroke was sufficient for neuroprotection. TLR4 can induce both IFNbeta and interferon-stimulated genes through its adapter molecule Toll/interleukin receptor domain-containing adaptor-inducing IFNbeta (TRIF) and the IRF3 transcription factor. We show in oxygen glucose deprivation of cortical neurons, an in vitro model of stroke, that activation of TRIF after stroke reduces neuronal death. Furthermore, mice lacking IRF3 were not protected by LPS preconditioning in our in vivo model. Our studies constitute the first demonstration of the neuroprotective capacity of TRIF/IRF3 signaling and suggest that interferon-stimulated genes, whether induced by IFNbeta or by enhanced TLR signaling to IRF3, are a potent means of protecting the brain against ischemic damage.

Interaction of NG2(+) glial progenitors and microglia/macrophages from the injured spinal cord.

Spinal cord contusion produces a central lesion surrounded by a peripheral rim of residual white matter. Despite stimulation of NG2(+) progenitor cell proliferation, the lesion remains devoid of normal glia chronically after spinal cord injury (SCI). To investigate potential cell-cell interactions of the predominant cells in the lesion at 3 days after injury, we used magnetic activated cell sorting to purify NG2(+) progenitors and OX42(+) microglia/macrophages from contused rat spinal cord. Purified NG2(+) cells from the injured cord grew into spherical masses when cultured in defined medium with FGF2 plus GGF2. The purified OX42(+) cells did not form spheroids and significantly reduced sphere growth by NG2(+) cells in co-cultures. Conditioned medium from these OX42(+) cells, unlike that from normal peritoneal macrophages or astrocytes also inhibited growth of NG2(+) cells, suggesting inhibition by secreted factors. Expression analysis of freshly purified OX42(+) cells for a panel of six genes for secreted factors showed expression of several that could contribute to inhibition of NG2(+) cells. Further, the pattern of expression of four of these, TNFalpha, TSP1, TIMP1, MMP9, in sequential coronal tissue segments from a 2 cm length of cord centered on the injury epicenter correlated with the expression of Iba1, a marker gene for OX42(+) cells, strongly suggesting a potential regional influence by activated microglia/macrophages on NG2(+) cells in vivo after SCI. Thus, the nonreplacement of lost glial cells in the central lesion zone may involve, at least in part, inhibitory factors produced by microglia/macrophages that are concentrated within the lesion.

TLR9 bone marrow chimeric mice define a role for cerebral TNF in neuroprotection induced by CpG preconditioning.

Systemic preconditioning with the TLR9 ligand CpG induces neuroprotection against brain ischemic injury through a tumor necrosis factor (TNF)-dependent mechanism. It is unclear how systemic administration of CpG engages the brain to induce the protective phenotype. To address this, we created TLR9-deficient reciprocal bone marrow chimeric mice lacking TLR9 on either hematopoietic cells or radiation-resistant cells of nonhematopoietic origin. We report that wild-type mice reconstituted with TLR9-deficient hematopoietic cells failed to show neuroprotection after systemic CpG preconditioning. Further, while hematopoietic expression of TLR9 is required for CpG-induced neuroprotection it is not sufficient to restore protection to TLR9-deficient mice that are reconstituted with hematopoietic cells bearing TLR9. To determine whether the absence of protection was associated with TNF, we examined TNF levels in the systemic circulation and the brain. We found that although TNF is required for CpG preconditioning, systemic TNF levels did not correlate with the protective phenotype. However, induction of cerebral TNF mRNA required expression of TLR9 on both hematopoietic and nonhematopoietic cells and correlated with neuroprotection. In accordance with these results, we show the therapeutic potential of intranasal CpG preconditioning, which induces brain TNF mRNA and robust neuroprotection with no concomitant increase in systemic levels of TNF.

Inhibition of Factor XII-Mediated Activation of Factor XI Provides Protection Against Experimental Acute Ischemic Stroke in Mice.

Blood coagulation factor XI (FXI) is an established risk factor for acute ischemic stroke (AIS) and thrombosis, but is also needed for normal hemostasis. Contact factor XII (FXII), an upstream activator of FXI, also contributes to experimental stroke, but is not required for hemostasis. We investigated whether selectively inhibiting FXII-mediated FXI activation, while leaving other FXI and FXII functions intact, could improve the outcome of experimental AIS in mice. Twenty-four hours before induction of AIS by placement of a filament into the internal carotid artery for 60 min, mice were anticoagulated with an antibody directed against the apple 2 domain of FXI. This antibody selectively reduces the prothrombotic activation of FXI by FXIIa but does not affect activated FXI or hemostatic activation of FXI by thrombin, thus leaving hemostasis intact in mice and primates. In this model of AIS/reperfusion injury, mice that received the antibody before AIS displayed less ischemic damage, manifested as reduced cerebral infarction and fibrin deposition (thrombosis), increased cortical reperfusion, and improved neurological behavior. Further, the antibody-anticoagulated mice had no detectable hemostasis impairment. Consistent with the neuroprotective phenotype of FXII-deficient mice, our data suggest that a single molecular event, FXII-mediated FXI activation, contributes to the development of experimental AIS.

It's all in the family: multiple Toll-like receptors offer promise as novel therapeutic targets for stroke neuroprotection.

Ischemic tolerance is a biological process that can be utilized to unlock the brain's own endogenous protection mechanisms and, as such, holds true promise for patients at risk of ischemic injury. Experimentally, preconditioning with various Toll-like receptor (TLR) agonists has now been demonstrated to successfully attenuate ischemic damage, partly through genomic reprogramming of the body's response to stroke. This treatment diminishes the inflammatory response to stroke and at the same time enhances the production of anti-inflammatory cytokines and neuroprotective mediators. This review discusses recent discoveries about the role of TLRs in preconditioning and ischemic tolerance.

Comparison of the effects of complete and incomplete spinal cord injury on lower urinary tract function as evaluated in unanesthetized rats.

In rats, phasic external urethral sphincter (EUS) activity (bursting) is postulated to be crucial for efficient voiding. This has been reported to be lost after spinal cord transection (txSCI), contributing to impaired function. However, anesthesia may confound evaluating EUS activity. We therefore evaluated urodynamic parameters in unanesthetized, restrained rats and compared the effects of txSCI to that of a clinically relevant, incomplete, contusive injury (iSCI) on lower urinary tract function. Adult female rats were subjected to txSCI or standardized iSCI at the T8 vertebral level. As expected, all injured rats were initially unable to void but developed a reflex bladder with time, with iSCI rats recovering more rapidly than txSCI rats. LUT function was evaluated urodynamically at 2 and 6 weeks after injury. In response to infusion of saline into the bladder, controls consistently exhibited coordinated contraction of the bladder and activation of the EUS in a phasic pattern and had a high voiding efficiency (86.4+/-2.5%). Voiding efficiency of iSCI rats was reduced to approximately 57% and txSCI rats to approximately 32%. However, two different patterns of EUS activity during voiding were present in both txSCI and iSCI groups at both time points: (1) rats with phasic EUS activity, similar to controls and (2) those that only exhibited tonic EUS activity during voiding. The former had more normal voiding efficiencies. Thus, phasic EUS activity and the improved voiding efficiency associated with it can occur and can be detected in unanesthetized rats after both incomplete and complete SCI.

Local and distal responses to injury in the rapid functional recovery from spinal cord contusion in rat pups.

Young rats display an accelerated rate of locomotor recovery after contusive spinal cord injury (SCI) compared to adults subjected to a similar standardized injury. We examined possible differences in the responses to SCI at the injury site and in the distal cord that might contribute to this rapid recovery. P14-15 rats were studied at 1, 3, 5, 7, and 28 days after injury at T8 produced with a weight drop device (10 g x 2.5 cm). We used immunohistochemistry to investigate distal plasticity of serotonergic and noradrenergic pathways that have been shown to modulate locomotion. After SCI, pups exhibited an expected decrease in monoaminergic innervation of the lumbosacral cord, consistent with partial loss of these descending pathways. Unlike published results for the adult, we found no evidence of partial reinnervation with time after injury. On the other hand, oligodendrocytes at and near the lesion epicenter of the young rats appeared unexpectedly resilient to the insult. No evidence of oligodendrocyte loss in spared white matter was detected at 24 h after injury, as compared to the 50% loss reported in adults. Rather, there was a significant increase in the density of oligodendrocytes by 5 days after injury that was associated with a dramatic upregulation of markers for glial progenitor cells after pup SCI. Our results suggest that an altered glial response near the injury epicenter as compared to that in adults is likely to contribute to the more rapid rate of recovery in hindlimb locomotor function in young rats after SCI.