Thrombin Preconditioning Research Articles

Thrombin Preconditioning Improves the Therapeutic Efficacy of Mesenchymal Stem Cells in Severe Intraventricular Hemorrhage Induced Neonatal Rats.

Severe intraventricular hemorrhage (IVH) remains a major cause of high mortality and morbidity in extremely preterm infants. Mesenchymal stem cell (MSC) transplantation is a possible therapeutic option, and development of therapeutics with enhanced efficacy is necessary. This study investigated whether thrombin preconditioning improves the therapeutic efficacy of human Wharton’s jelly-derived MSC transplantation for severe neonatal IVH, using a rat model. Severe neonatal IVH was induced by injecting 150 μL blood into each lateral ventricle on postnatal day (P) 4 in Sprague-Dawley rats. After 2 days (P6), naïve MSCs or thrombin-preconditioned MSCs (1 × 105/10 μL) were transplanted intraventricularly. After behavioral tests, brain tissues and cerebrospinal fluid of P35 rats were obtained for histological and biochemical analyses, respectively. Thrombin-preconditioned MSC transplantation significantly reduced IVH-induced ventricular dilatation on in vivo magnetic resonance imaging, which was coincident with attenuations of reactive gliosis, cell death, and the number of activated microglia and levels of inflammatory cytokines after IVH induction, compared to naïve MSC transplantation. In the behavioral tests, the sensorimotor and memory functions significantly improved after transplantation of thrombin-preconditioned MSCs, compared to naïve MSCs. Overall, thrombin preconditioning significantly improves the therapeutic potential and more effectively attenuates brain injury, including progressive ventricular dilatation, gliosis, cell death, inflammation, and neurobehavioral functional impairment, in newborn rats with induced severe IVH than does naïve MSC transplantation.

Thrombin Preconditioning Boosts Biogenesis of Extracellular Vesicles from Mesenchymal Stem Cells and Enriches Their Cargo Contents via Protease-Activated Receptor-Mediated Signaling Pathways.

We investigated the role of protease-activated receptor (PAR)-mediated signaling pathways in the biogenesis of human umbilical cord blood-derived mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) and the enrichment of their cargo content after thrombin preconditioning. Immunoblot analyses showed that MSCs expressed two PAR subtypes: PAR-1 and PAR-3. Thrombin preconditioning significantly accelerated MSC-derived EV biogenesis more than five-fold and enriched their cargo contents by more than two-fold via activation of Rab5, early endosomal antigen (EEA)-1, and the extracellular signal regulated kinase (ERK)1/2 and AKT signaling pathways. Blockage of PAR-1 with the PAR-1-specific antagonist, SCH79797, significantly suppressed the activation of Rab5, EEA-1, and the ERK1/2 and AKT pathways and subsequently increased EV production and enriched EV cargo contents. Combined blockage of PAR-1 and PAR-3 further and significantly inhibited the activation of Rab5, EEA-1, and the ERK1/2 and AKT pathways, accelerated EV production, and enriched EV cargo contents. In summary, thrombin preconditioning boosted the biogenesis of MSC-derived EVs and enriched their cargo contents largely via PAR-1-mediated pathways and partly via PAR-1-independent, PAR-3-mediated activation of Rab5, EEA-1, and the ERK1/2 and AKT signaling pathways.

Thrombin Preconditioning Enhances Therapeutic Efficacy of Human Wharton's Jelly-Derived Mesenchymal Stem Cells in Severe Neonatal Hypoxic Ischemic Encephalopathy.

We investigated whether thrombin preconditioning of human Wharton’s jelly–derived mesenchymal stem cells (MSCs) improves paracrine potency and thus the therapeutic efficacy of naïve MSCs against severe hypoxic ischemic encephalopathy (HIE). Thrombin preconditioning significantly enhances the neuroprotective anti-oxidative, anti-apoptotic, and anti-cytotoxic effects of naïve MSCs against oxygen–glucose deprivation (OGD) of cortical neurons in vitro. Severe HIE was induced in vivo using unilateral carotid artery ligation and hypoxia for 2 h and confirmed using brain magnetic resonance imaging (MRI) involving >40% of ipsilateral hemisphere at postnatal day (P) 7 in newborn rats. Delayed intraventricular transplantation of 1 × 105 thrombin preconditioned but not naïve MSCs at 24 h after hypothermia significantly enhanced observed anti-inflammatory, anti-astroglial, and anti-apoptotic effects and the ensuing brain infarction; behavioral tests, such as cylinder rearing and negative geotaxis tests, were conducted at P42. In summary, thrombin preconditioning of human Wharton’s jelly-derived MSCs significantly boosted the neuroprotective effects of naïve MSCs against OGD in vitro by enhancing their anti-oxidative, anti-apoptotic, and anti-cytotoxic effects, and significantly attenuated the severe HIE-induced brain infarction and improved behavioral function tests in vivo by maximizing their paracrine anti-inflammatory, anti-astroglial, and anti-apoptotic effects.

Thrombin Preconditioning of Extracellular Vesicles Derived from Mesenchymal Stem Cells Accelerates Cutaneous Wound Healing by Boosting Their Biogenesis and Enriching Cargo Content.

The aim of this study was to determine the optimal preconditioning regimen for the wound healing therapeutic efficacy of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs). To this end, we compared various preconditioning regimens for both the quantitative and qualitative production of MSC-derived EVs, and their therapeutic efficacy for proangiogenic activity in vitro and cutaneous wound healing in vivo. After preconditioning with thrombin (40 U), H2O2 (50 μM), lipopolysaccharide (1 μg/mL), or hypoxia (10% O2), EV secretion was assessed quantitatively by measuring production per cell and protein quantification, and qualitatively by measuring a proteome profiler and an enzyme-linked immunosorbent assay (ELISA) contained within EVs. The therapeutic efficacy of EVs was assessed in vitro by proliferation, migration and tube formation assays of human umbilical cord blood endothelial cells (HUVECs), and in vivo by quantification of cutaneous wound healing. Thrombin preconditioning optimally boosted EV production and enriched various growth factors including vascular endothelial growth factor and angiogenin contained within EVs compared to other preconditioning regimens. Thrombin preconditioning optimally enhanced proliferation, the migration and tube formation of HUVECs in vitro via pERK1/2 and pAKT signaling pathways, and cutaneous wound healing in vivo compared to other preconditioning regimens. Thrombin preconditioning exhibited optimal therapeutic efficacy compared with other preconditioning regimens in promoting proangiogenic activity in vitro and in enhancing cutaneous wound healing in vivo. These preconditioning regimen-dependent variations in therapeutic efficacy might be mediated by boosting EV production and enriching their cargo content.

Increase of aquaporin 9 expression in astrocytes participates in astrogliosis.

Here we assess the potential functional role of increased aquaporin 9 (APQ9) in astrocytes. Increased AQP9 expression was achieved in primary astrocyte cultures by transfection of a plasmid-containing green fluorescent protein fused to either wild-type or mutated human AQP9. Increased AQP9 expression and phosphorylation at Ser222 were associated with a significant change in astrocyte morphology, mainly with a higher number of processes. Similar phenotypic changes are observed in astrogliosis processes after injury. In parallel, we observed that in vivo, thrombin preconditioning before ischemic stroke induced an early increase in AQP9 expression in the male mouse brain. This increased AQP9 expression was also associated with astrocyte morphological changes, especially in the white matter tract. Astrocyte reactivity is debated as being either beneficial or deleterious. As thrombin preconditioning leads to a decrease in lesion size after stroke, our data suggest that the early increase in AQP9 concomitant with astrocyte reactivity leads to a beneficial effect. © 2017 Wiley Periodicals, Inc.

Effect of thrombin preconditioning on migration of subventricular zone-derived cells after intracerebral hemorrhage in rats

Objective: To investigate the effect of thrombin preconditioning (TPC) on the intracerebral hemorrhage (ICH)-induced proliferation, migration, and function of subventriclular zone (SVZ) cells and to find new strategies that enhance endogenous neurogenesis after ICH.Methods: Male Sprague-Dawley rats were randomly divided into 3 groups (ICH, TPC, and control group). Rats of each group were randomly divided into 5 subgroups (3-d, 7-d, 14-d, 21-d, and 28-d subgroup). ICH was caused by intrastrial stereotactic administration of collagenase type IV. Brdu was used to label newborn SVZ cells. Organotypic brain slices were cultured to dynamically observe the migration of SVZ cells at living brain tissue. Migration of Dil-labeled SVZ cells in living brain slices was traced by time-lapse microscopy. To assess whether SVZ cells migrating to injured striatum had the ability to form synapses with other cells, brain slices from each group were double immunolabeled with Brdu and synapsin I.Results: The number of Brdu-positive cells markedly increased in the ipsilateral SVZ and striatum 3 days after TPC, peaked at 14 days (P < 0.01), continued to 21 days, and then gradually decreased at 28 days with significant difference compared to the ICH group at each time point (P < 0.01). Migration of Dil-labeled SVZ cells in brain slices in each group was observed and imaged during a 12-h period. Dil-labeled SVZ cells in the TPC group were observed to migrate laterally toward striatum with time with a faster velocity compared to the ICH group (P < 0.01). Our study also demonstrated that TPC induced strong colocalization of Brdu and synapsin I in the ipsilateral striatum between 3 and 28 days after injury.TPC made colocalization of Brdu and synapsin I appear earlier and continue for a longer time compared to the ICH group.Conclusions: Our results demonstrated that TPC could promote proliferation, migration, and function of SVZ cells after ICH, which may provide a new idea for enhancing endogenous neurogenesis and developing new therapeutic strategies against ICH-induced brain injury.

Thrombin Preconditioning in Surgical Brain Injury in Rats.

The surgical brain injury model replicates neurosurgical brain parenchymal damage. Postsurgical brain edema correlates with postoperative neurological dysfunction. Intranasal administration is a proven method of delivering therapies to brain tissue. Thrombin preconditioning decreased brain edema and improved neurological outcomes in models of ischemic brain injury. We hypothesized thrombin preconditioning in surgical brain injury may improve postoperative brain edema and neurological outcomes. Adult male Sprague-Dawley rats (n = 78) weighing 285-355 g were randomly assigned to sham or pre-injury treatment: one-time pretreatment 1 day prior, one-time pretreatment 5 days prior, and daily preconditioning for 5 days prior. Treatment arms were divided into vehicle or thrombin therapies, and subdivided into intranasal (thrombin 5 units/50 μL 0.9 % saline) or intracerebral ventricular (thrombin 0.1 unit/10 μL 0.9 % saline) administration. Blinded observers performed neurological testing 24 h after brain injury followed immediately by measurement of brain water content. There was a significant difference in ipsilateral brain water content and neurological outcomes between all treatment groups and the sham group. However, there was no change in brain water content or neurological outcomes between thrombin- and vehicle-treated animals. Thrombin preconditioning did not significantly improve brain edema or neurological function in surgical brain injury in rats.

Endogenous Protease Nexin-1 Protects against Cerebral Ischemia

The serine protease thrombin plays a role in signalling ischemic neuronal death in the brain. Paradoxically, endogenous neuroprotective mechanisms can be triggered by preconditioning with thrombin (thrombin preconditioning, TPC), leading to tolerance to cerebral ischemia. Here we studied the role of thrombin’s endogenous potent inhibitor, protease nexin-1 (PN-1), in ischemia and in tolerance to cerebral ischemia induced by TPC. Cerebral ischemia was modelled in vitro in organotypic hippocampal slice cultures from rats or genetically engineered mice lacking PN-1 or with the reporter gene lacZ knocked into the PN-1 locus PN-1HAPN-1-lacZ/HAPN-1-lacZ (PN-1 KI) exposed to oxygen and glucose deprivation (OGD). We observed increased thrombin enzyme activity in culture homogenates 24 h after OGD. Lack of PN-1 increased neuronal death in the CA1, suggesting that endogenous PN-1 inhibits thrombin-induced neuronal damage after ischemia. OGD enhanced β-galactosidase activity, reflecting PN-1 expression, at one and 24 h, most strikingly in the stratum radiatum, a glial cell layer adjacent to the CA1 layer of ischemia sensitive neurons. TPC, 24 h before OGD, additionally increased PN-1 expression 1 h after OGD, compared to OGD alone. TPC failed to induce tolerance in cultures from PN-1−/− mice confirming PN-1 as an important TPC target. PN-1 upregulation after TPC was blocked by the c-Jun N-terminal kinase (JNK) inhibitor, L-JNKI1, known to block TPC. This work suggests that PN-1 is an endogenous neuroprotectant in cerebral ischemia and a potential target for neuroprotection.

Effect of Iron Chelators on Methemoglobin and Thrombin Preconditioning

Cell loss immediately adjacent to an intracerebral hemorrhage may be mediated in part by the toxicities of extracellular hemoglobin (Hb) and thrombin. However, at low concentrations, these proteins induce tolerance to hemin and iron that may limit further peri-hematomal injury as erythrocyte lysis progresses. The mechanisms mediating these preconditioning effects have not been completely defined, but increased expression of both heme oxygenase (HO)-1 and iron binding proteins likely contributes. In the present study, we hypothesized that iron chelator therapy would attenuate this protective response. Pretreatment of cortical glial cultures (> 90 % GFAP+) with 3 μM methemoglobin (metHb) or 5 units/ml thrombin for 24 h was nontoxic per se, and increased HO-1 and ferritin expression. When challenged with a toxic concentration of hemin, the increase in cellular redox-active iron was attenuated in preconditioned cultures and cell survival was increased. However, if cultures were pretreated with metHb or thrombin plus deferoxamine or 2,2'-bipyridyl, ferritin induction was prevented and cellular redox-active iron increased with hemin treatment. Preconditioning-mediated cytoprotection was consistently reduced by deferoxamine, while 2,2'-bipyridyl had a variable effect. Neither chelator altered HO-1 expression. A cytoprotective response was preserved when chelator therapy was limited to 11 hours of the 24 h preconditioning interval. These results suggest a potentially deleterious effect of continuous iron chelator therapy after ICH. Intermittent therapy may remove peri-hematomal iron without negating the benefits of exposure to low concentrations of Hb or thrombin.

Thrombin-induced neuronal protection: Role of the mitogen activated protein kinase/ribosomal protein S6 kinase pathway

Our previous studies have found that intracerebral pretreatment with a low dose of thrombin (thrombin preconditioning, TPC) reduces infarct volume and attenuates brain edema after focal cerebral ischemia. In this study, we examined whether TPC protects against the neuronal death induced by oxygen glucose deprivation (OGD), and whether the protection is through thrombin receptors and the p44/42 mitogen activated protein kinases (MAPK)/ribosomal protein S6 kinases (p70 S6K) pathway. Expression of protease-activated receptors (PARs) mRNA was detected in cultured primary rat neurons and thrombin upregulated PAR-1 and PAR-4 mRNA expression. TPC reduced OGD-induced neuronal death (e.g. dead cells: 52.5 ± 5.4% vs. 72.3 ± 7.2% in the control group, n = 6, p < 0.01). Agonists of PAR-1 and PAR-4 mimicked the effects of thrombin and reduced OGD-induced neuronal death. Pretreatment with thrombin or PAR agonists induced the upregulation of activated p44/42 MAPK and p70S6K (Thr 421/Ser 424). PD98059, an inhibitor of p44/42 MAPK kinase, blocked thrombin-induced upregulation of activated p44/42 MAPK and p70S6K. It also reduced TPC-induced neuronal protection (e.g. dead cells: 68.2 ± 5.2% vs. 56.9 ± 4.6% in vehicle + TPC group, n = 6, p < 0.05). These results suggest that TPC-induced ischemic tolerance is through activation of thrombin receptors and the p44/42 MAPK/p70S6K pathway.

Activation of c‐Jun in the nuclei of neurons of the CA‐1 in thrombin preconditioning occurs via PAR‐1

Recently it has been shown that the c-Jun N-terminal kinase (JNK) plays a role in thrombin preconditioning (TPC) in vivo and in vitro. To investigate further the pathways involved in TPC, we performed an immunohistochemical study in hippocampal slice cultures. Here we show that the major target of JNK, the AP-1 transcription factor c-Jun, is activated by phosphorylation in the nuclei of neurons of the CA1 region by using phospho-specific antibodies against the two JNK phosphorylation sites. The activation is early and transient, peaking at 90 min and not present by 3 hr after low-dose thrombin administration. Treatment of cultures with a synthetic thrombin receptor agonist results in the same c-Jun activation profile and protection against subsequent OGD, both of which are prevented by specific JNK inhibitors, showing that thrombin signals through PAR-1 to JNK. By using an antibody against the Ser 73 phosphorylation site of c-Jun, we identify possible additional TPC substrates.

Protective Role of Early Aquaporin 4 Induction against Postischemic Edema Formation

Aquaporin 4 (AQP4) is a water channel involved in water movements across the cell membrane and is spatially organized on the cell surface in orthogonal array particles (OAPs). Its role in edema formation or resolution after stroke onset has been studied mainly at late time points. We have shown recently that its expression is rapidly induced after ischemia coinciding in time with an early swelling of the ischemic hemisphere. There are two isoforms of AQP4: AQP4-M1 and AQP4-M23. The ratio of these isoforms influences the size of the OAPs but the functional impact is not known. The role of the early induction of AQP4 is not yet known. Thrombin preconditioning in mice provides a useful model to study endogenous protective mechanisms. Using this model, we provide evidence for the first time that the early induction of AQP4 may contribute to limit the formation of edema and that the AQP4-M1 isoform is predominantly induced in the ischemic tissue at this time point. Although it prevents edema formation, the early induction of the AQP4 expression does not prevent the blood-brain barrier disruption, suggesting an effect limited to the prevention of edema formation possibly by removing of water from the tissue.

Prothrombin/thrombin and the thrombin receptors PAR-1 and PAR-4 in the brain: Localization, expression and participation in neurodegenerative diseases

Emerging evidence demonstrates that thrombin exerts physiological and pathological functions in the central nervous system. Both prothrombin and its active form thrombin have been detected locally in the brain. The cellular functions of thrombin are mainly regulated by G protein-coupled protease-activated receptors (PARs). Thrombin can signal via PAR-1, PAR-3 and PAR-4. Some neurological diseases (e.g. Alzheimer's disease or Parkinson's disease) are characterized by increased levels of both active thrombin and PAR-1. This indicates that thrombin and its receptor may be closely involved in the development of neurodegenerative processes. The role of thrombin in brain injury can be either protective or deleterious, depending on the concentration of thrombin. Thrombin at high concentrations exacerbates brain damage. In contrast, low concentrations of thrombin rescue neural cells from death after brain insults. Also thrombin preconditioning has neuroprotective effects. Therefore, thrombin and thrombin receptors represent novel therapeutic targets for treating neurodegenerative diseases.

The effect of thrombin on a 6-hydroxydopamine model of Parkinson's disease depends on timing

Recent results in animal models suggest that thrombin may modulate brain injury in Parkinson's disease (PD). High doses of thrombin (∼20 U) can damage dopaminergic neurons, while we have found that low dose thrombin (1 U), given several days before a brain insult (thrombin preconditioning), is protective in models of PD and stroke. However, the effects of such low levels of thrombin at the time of, or after, exposure to the dopamine neurotoxin 6-hydroxydopamine (6-OHDA) have not been examined and are the focus of this study. In the first set of experiments, rats received co-administration of thrombin (1 U) or saline and 6-OHDA (5 μg) into the medial forebrain bundle. 6-OHDA + thrombin resulted in striking increases in behavioral deficits, compared to 6-OHDA + saline. Similarly, co-administration of an agonist to protease-activated receptor (PAR)-1, a thrombin receptor, also resulted in significantly greater behavioral deficits. In a second set of experiments, thrombin (1 U) or saline was administered 1 or 7 days after 6-OHDA to determine the effects of thrombin after 6-OHDA. Surprisingly, the rats that received saline had strikingly increased behavioral and neurochemical deficits resulting from the 6-OHDA lesion, while delayed thrombin administration prevented this effect. The results indicate that thrombin has differential effects in the 6-OHDA model, dependent on the time of administration. The ability of a second cannula insertion with saline infusion to increase dramatically deficits raises questions as to what role physical injury to already susceptible cells might play in the pathogenesis of some cases of PD.

Thrombin-induced ischemic tolerance is prevented by inhibiting c-jun N-terminal kinase

We have studied ischemic tolerance induced by the serine protease thrombin in two different models of experimental ischemia. In organotypic hippocampal slice cultures, we demonstrate that incubation with low doses of thrombin protects neurons against a subsequent severe oxygen and glucose deprivation. l-JNKI1, a highly specific c-jun N-terminal kinase (JNK) inhibitor, and a second specific JNK inhibitor, SP600125, prevented thrombin preconditioning (TPC). We also show that the exposure to thrombin increases the level of phosphorylated c-jun, the major substrate of JNK. TPC, in vivo, leads to significantly smaller lesion sizes after a 30-min middle cerebral artery occlusion (MCAo), and the preconditioned mice were better off in the three tests used to evaluate functional recovery. In accordance with in vitro results, TPC in vivo was prevented by administration of l-JNKI1, supporting a role for JNK in TPC. These results, from two different TPC models and with two distinct JNK inhibitors, show that JNK is likely to be involved in TPC.

Protease-activated receptor-1 mediates protection elicited by thrombin preconditioning in a rat 6-hydroxydopamine model of Parkinson's disease

The etiology of Parkinson's disease remains poorly understood, and current treatment options do not slow disease progression. Recently, chemical (thrombin) preconditioning (TPC) was found to be protective in a 6-hydroxydopamine (6-OHDA) model of the disease. It is important to understand the mechanisms behind these thrombin-induced protective effects. The current study was conducted in the rat to determine whether the protective effects of TPC are mediated via activation of protease-activated receptors (PARs). Preconditioning with specific local infusion of agonist peptides for PAR-1 and PAR-4 3 days before unilateral 6-OHDA administration (10 μg into the medial forebrain bundle) was tested. In addition, co-administration of a PAR-1 antagonist with TPC was examined. In a neurobehavioral assessment battery, PAR-1 agonist preconditioning provided protection in a vibrissae-elicited forelimb placing test, a forelimb-use asymmetry test, and a corner turn test. In addition, inclusion of a PAR-1 antagonist prevented the protective effects elicited by TPC. In contrast to the effects of the PAR-1 agonist, PAR-4 agonist preconditioning afforded no such protection. Indeed, in a lower-dose model of 6-OHDA (5 μg), PAR-4 preconditioning significantly increased behavioral deficits. These results indicate that the protective effects of TPC in this model are mediated through PAR-1 activation. Neither the effects of PAR-1 nor TPC on later 6-OHDA-induced behavioral deficits appeared to be mediated through (DA) content sparing. Further mechanistic studies on the actions of PAR-1 and PAR-4 as detrimental in experimental models of Parkinson's disease are warranted.

Thrombin preconditioning provides protection in a 6-hydroxydopamine Parkinson's disease model

Low-dose thrombin given several days before lesioning is neuroprotective in ischemic and hemorrhagic models of stroke, an effect termed thrombin preconditioning (TPC). Here, the ability of TPC to provide protection in a 6-hydroxydopamine (6-OHDA) model of Parkinson's disease (PD) was evaluated. All animals received 10 μg 6-OHDA into the right medial forebrain bundle. Three days prior to 6-OHDA, the animals received either 1 U rat thrombin ( n = 17) or saline ( n = 14) 1 mm above the site of neurotoxin delivery. The animals were then evaluated for neurobehavioral deficits until 21 days post-injection. TPC animals performed significantly better on both a vibrissae-elicited forelimb placing test and a forelimb-use asymmetry test than the saline controls. The animals were then sacrificed for either catecholamine determination by HPLC with electrochemical detection or for histopathology to determine lateral ventricular volume or striatal tyrosine hydroxylase immunoreactivity. Although TPC did not protect against the dopamine depletion associated with this severe model, it did reduce dopaminergic terminal loss and ventricular enlargement as compared to saline-treated animals. This report presents the new finding that preconditioning (and TPC in particular) provides protection in a 6-OHDA PD model. Understanding the mechanisms involved in TPC-mediated protection may stimulate innovative therapeutic regimens.

Thrombin preconditioning attenuates brain edema induced by erythrocytes and iron.

Pretreatment with a low intracerebral dose of thrombin reduces brain edema after hemorrhagic and thrombo-embolic stroke. We have termed this phenomena thrombin preconditioning (TPC) or thrombin-induced brain tolerance. Red blood cell lysis and iron overload contribute to delayed edema formation after intracerebral hemorrhage. The present study examined whether TPC can attenuate the brain edema induced by lysed red blood cells or iron. It also examined whether TPC is associated with increasing hypoxia inducible factor-1alpha (HIF-1alpha) levels and alterations in two HIF-1alpha target genes, transferrin (Tf) and transferrin receptor (TfR), within the brain. Brain edema was measured by wet/dry weight method. HIF-1alpha, Tf, and TfR were measured by Western blot analysis and immunohistochemistry. We found that TPC reduces the edema induced by infusion of lysed red blood cells and iron. Thrombin increases HIF-1alpha levels through p44/42 mitogen activated protein kinases pathway. Thrombin also increases Tf and TfR levels in the brain. These results indicate that HIF-1alpha and its target genes may be involved in thrombin-induced brain tolerance.

Nestin expression after experimental intracerebral hemorrhage

The current study examines nestin expression after intracerebral hemorrhage (ICH), the role of different blood components in nestin upregulation, and the possibility that low doses of thrombin that induce tolerance to brain injury (thrombin preconditioning) might also induce nestin expression. Adult male Sprague–Dawley rats received an intracaudate injection of either whole blood, thrombin (1 or 5 U) or red blood cells (RBCs). Animals were sacrificed for single and double labeling immunohistochemistry to identify which cells express nestin, and for Western blotting to quantify nestin expression. By immunohistochemistry, nestin immunoreactivity was present in large numbers of astrocytes, surrounding the hematoma from day 3 to 1 week after ICH. After 2 weeks, nestin immunoreactivity was co-localized with a neuronal marker (neuronal specific enolase). By Western blot analysis, nestin was strongly expressed at day 3 ( P<0.01) and 1 week ( P<0.01), and expression persisted for at least 1 month ( P<0.05). Intracerebral injection of thrombin or lysed RBCs resulted in a marked increase in nestin expression. Interestingly, injection of a low dose of thrombin that induces brain tolerance also upregulated nestin. The ICH-induced nestin expression in astrocytes may reflect an early response of these cells to injury, while the delayed expression in neurons might be a part of the adaptative response to injury perhaps leading to recovery of function. Nestin induction by a low dose of thrombin suggests that specific receptor-mediated pathways are involved in inducing nestin expression and that nestin may play a role in thrombin preconditioning.

Thrombin preconditioning upregulates transferrin and transferrin receptor and reduces brain edema induced by lysed red blood cells.

Pretreatment with a low dose of thrombin reduces brain edema after both hemorrhagic and ischemic stroke. We call this phenomenon thrombin preconditioning (TPC) or thrombin-induced brain tolerance. The present study examines whether TPC can attenuate the brain edema induced by lysed red blood cells (RBCs) to determine whether thrombin production early in an intracerebral hemorrhage (ICH) might alter potentially injurious events associated with clot resolution. It also examines whether TPC might be protective by altering iron handling within the brain, particularly through modulating transferrin (Tf) and transferrin receptor (TfR) levels. Brain edema was measured by wet/dry weight. Western blot analysis and immunohistochemistry were used for Tf and TfR measurements. We found that TPC reduces lysed RBC-induced brain edema and upregulates both Tf and TfR levels in the brain. Thrombin formation after an ICH may be part of a signaling cascade that acts to limit potentially injurious events associated with clot resolution through altering iron-handling proteins.