HA Mice Research Articles

Susceptibility to Pentylenetetrazole-Induced Seizures in Mice with Distinct Activity of the Endogenous Opioid System.

Currently, pharmacotherapy provides successful seizure control in around 70% of patients with epilepsy; however, around 30% of cases are still resistant to available treatment. Therefore, effective anti-epileptic therapy still remains a challenge. In our study, we utilized two mouse lines selected for low (LA) and high (HA) endogenous opioid system activity to investigate the relationship between down- or upregulation of the opioid system and susceptibility to seizures. Pentylenetetrazole (PTZ) is a compound commonly used for kindling of generalized tonic-clonic convulsions in animal models. Our experiments revealed that in the LA mice, PTZ produced seizures of greater intensity and shorter latency than in HA mice. This observation suggests that proper opioid system tone is crucial for preventing the onset of generalized tonic-clonic seizures. Moreover, a combination of an opioid receptor antagonist-naloxone-and a GABA receptor agonist-diazepam (DZP)-facilitates a significant DZP-sparing effect. This is particularly important for the pharmacotherapy of neurological patients, since benzodiazepines display high addiction risk. In conclusion, our study shows a meaningful, protective role of the endogenous opioid system in the prevention of epileptic seizures and that disturbances in that balance may facilitate seizure occurrence.

Additional Factor X Enhances Emicizumab-Driven Coagulation Function in Patients with Hemophilia A and Hemophilia A Mice.

Bypassing agents are used for breakthrough bleedings in patients with hemophilia A with inhibitor (PwHAwI) receiving emicizumab prophylaxis. Previous study demonstrated a weak binding affinity between emicizumab and factor (F)X (K d; 1.85 μM), and that this value was much greater than the plasma FX concentration (∼130 nM). We speculated that increased FX levels could enhance coagulation potential in emicizumab-treated patients with hemophilia A (PwHA). To investigate the relationship between FX concentrations and emicizumab-driven coagulation. Plasma FX (up to 1,040 nM) and emicizumab (50 µg/mL) were added to FVIII-deficient plasmas, and plasma-derived FX (520 nM) or recombinant (r)FVIIa (2.2 µg/mL) was added to plasmas from three emicizumab-treated PwHAwI. The adjusted maximum coagulation velocity (Ad|min1|) by clot waveform analysis and peak thrombin (PeakTh) by thrombin generation assay in them were evaluated. Emicizumab (3.0 mg/kg), human (h)FIX (100 IU/kg), and various doses of hFX (100-500 IU/kg) were intravenously administered to HA mice. Clotting time/clot formation time (CT/CFT) were assessed using rotational thromboelastometry, and blood loss was estimated by a tail-clip assay. The addition of FX to FVIII-deficient plasma with emicizumab increased Ad|min1| and PeakTh. The coagulation parameters in emicizumab-treated PwHAwI spiked with additional FX remained within the normal range as well as the additional rFVIIa. In animal models, hFX injection shortened the CT and CT + CFT. The shorter CT and CT + CFT, and the lower blood loss were evident after 200 or 500 IU/kg hFX administration, and those indices were comparable to those in wild-type mice. Supplementation with FX may improve emicizumab-driven hemostasis in PwHA.

Glycocalyx-targeted therapy ameliorates age-related arterial dysfunction.

Advanced age is accompanied by arterial dysfunction, as well as a diminished glycocalyx, which may be linked to reduced high molecular weight-hyaluronan (HMW-HA) synthesis. However, the impact of glycocalyx deterioration in age-related arterial dysfunction is unknown. We sought to determine if manipulations in glycocalyx properties would alter arterial function. Tamoxifen-induced hyaluronan synthase 2 (Has2) reduction was used to decrease glycocalyx properties. Three weeks post-tamoxifen treatment, glycocalyx thickness was lower in Has2 knockout compared to wild-type mice (P<0.05). Has2 reduction induced arterial dysfunction, demonstrated by impaired endothelium-dependent dilation (EDD) and elevated aortic stiffness (P<0.05). To augment glycocalyx properties, old mice received 10 weeks of a glycocalyx-targeted therapy via Endocalyx™ (old+ECX), which contains HMW-HA and other glycocalyx components. Compared to old control mice, glycocalyx properties and EDD were augmented, and aortic stiffness decreased in old+ECX mice (P<0.05). Old+ECX mice had a more youthful aortic phenotype, demonstrated by lower collagen content and higher elastin content than old control mice (P<0.05). Functional outcomes were repeated in old mice that underwent a diet supplemented solely with HMW-HA (old+HA). Compared to old controls, glycocalyx properties and EDD were augmented, and aortic stiffness was lower in old+HA mice (P<0.05). We did not observe any differences between old+HA and old+ECX mice (P>0.05). Has2 reduction phenocopies age-related arterial dysfunction, while 10 weeks of glycocalyx-targeted therapy that restores the glycocalyx also ameliorates age-related arterial dysfunction. These findings suggest that the glycocalyx may be a viable therapeutic target to ameliorate age-related arterial dysfunction.

The IgG-degrading enzyme, Imlifidase, restores the therapeutic activity of FVIII in inhibitor-positive hemophilia A mice.

Neutralizing anti-factor VIII (FVIII) antibodies, known as FVIII inhibitors, represent a major drawback of replacement therapy in persons with congenital hemophilia A (PwHA), rendering further infusions of FVIII ineffective. FVIII inhibitors can also appear in non-hemophilic individuals causing acquired hemophilia A (AHA). The use of non-FVIII bypassing agents in cases of bleeds or surgery in inhibitor-positive patients is complicated by the lack of reliable biological monitoring and increased thrombotic risk. Imlifidase (IdeS) is an endopeptidase that degrades human immunoglobulin G (IgG); it was recently approved for hyperimmune patients undergoing renal transplants. Here we investigated the ability of IdeS to eliminate FVIII inhibitors in vitro and in a model of inhibitor-positive HA mice. IdeS cleaved anti-FVIII plasma IgG from PwHA and AHA patients, and hydrolyzed recombinant human anti-FVIII IgG independently from their subclass or specificity for the A2, A3, C1 or C2 domains of FVIII. In HA mice passively immunized with recombinant human anti-FVIII IgG, IdeS restored the hemostatic efficacy of FVIII, as evidenced by the correction of the bleeding tendency. Our results provide the proof of concept for the transient removal of FVIII inhibitors by IdeS, thereby opening a therapeutic window for efficient FVIII replacement therapy in inhibitor-positive patients.

Desferoxamine protects against hemophilic arthropathy through the upregulation of HIF-1α-BNIP3 mediated mitophagy

AimsHemophilic arthropathy (HA) is a typically iron overload induced joint disease secondary to continuous joint bleeding, however, the exact role of iron chelators in HA has not been fully elucidated. In the present study, we investigated whether desferoxamine (DFO), an iron chelator, could limit the development of HA and the underlying mechanisms. Materials and methodsA HA mice model was established by needle puncture in the left knees of FVIII-deficient hemophilic mice. HA progression was evaluated at 8 weeks after DFO administration. Moreover, chondrocytes were treated with ferric ammonium citrate (FAC) to mimic iron overload in vitro. Modulating effect of DFO on iron overload induced oxidative stress, chondrocytes apoptosis and extracellular matrix (ECM) degradation and the role of HIF-1α-BNIP3 mediated mitophagy were examined. Key findingsWe found that DFO limited the development of HA and protected iron overload induced ECM degradation, chondrocytes apoptosis and oxidative stress. Besides chelating Fe2+, we found that HIF-1α-BNIP3 mediated mitophagy played important roles in the protective effect of DFO. HIF-1α inhibition suppressed chondrocytes mitophagy process and partly diminished the protective effect of DFO on chondrocytes iron overload. SignificanceIn conclusion, DFO could protect against HA development via HIF-1α-BNIP3 mediated mitophagy, suggesting DFO might be a potential therapeutic supplement for HA treatment.

An Antibody Targeting Human FV Promotes Thrombin Generation and Reduces Bleeding in a Hemophilic Mouse Model

Hemophilia is a bleeding disorder that results from a deficiency of factor VIII (Hemophilia A; HA) or factor IX (Hemophilia B; HB). Protein replacement therapy ameliorates bleeding but can result in the development of neutralizing antibodies. Innovative nonfactor therapies are in development for hemophilia which target anticoagulant proteins such as tissue factor pathway inhibitor (TFPI), antithrombin, and activated protein C (APC). While these approaches show promise, they influence multiple coagulation factors and pathways which could raise safety concerns. Considering its essential role in coagulation in enhancing thrombin generation (TG), we hypothesized that altering the functional properties of FV/FVa could be an attractive approach for HA and HB treatment. Activated FV (FVa) assembles with FXa on activated cellular surfaces and enhances the kinetics of TG by several orders of magnitude. Here we assess whether monoclonal antibodies targeting all forms of FV are effective in promoting TG in hemophilia using both in vitro and in vivo models in hemophilic mice. Using an initial TG assay screen, we identified a procoagulant antibody (GB5) that binds with high affinity (Kd <0.5 nM) to human (h)FV, FVa, and FV-short and investigated its mechanism of action. Using proteolytic fragments of FV and western blotting experiments, we found that GB5 likely binds in the middle of the light chain region. To evaluate the functional effects of GB5 in vitro, we first employed a plasma-based TG assay using HA or HB plasma triggered with low (0.2 pM) tissue factor. The presence of saturating GB5 increased TG about 3-fold in HA plasma. Similar results were observed with HB plasma. Interestingly, when TG was essentially eliminated in HA and HB plasma with addition of exogenous APC, GB5 restored TG. These results suggest that the procoagulant effect of GB5 neutralizes the anticoagulant effect of APC and enhanced TG in hemophilic plasma To begin to assess the mechanism, the effect of GB5 on FV activation in the TG assay was evaluated. TG assay samples were removed over time, quenched, and analyzed by immunoblotting using labeled FV specific antibodies. In the presence of GB5, ~95% of the starting full-length FV was converted to FVa within 3 min compared to 5 min in the absence of GB5. These results show that FV is activated faster in the presence of GB5 in the TG assay. To extend these data, we next assessed the influence of GB5 in the TG assays using FV-deficient plasma supplemented with either FV or FVa and TFPI⍺. The data show that GB5 had essentially no effect on the TG assay when FV deficient plasma was reconstituted with FVa, but enhanced TG at least 3-fold when supplemented with FV. Consistent with this, the kinetics of prothrombin activation with FXa-FVa-membranes in the presence or absence of GB5 were the same. These data suggest that GB5 impacts reactions that influence FV, with no direct effect on FVa procoagulant function. To evaluate whether GB5 has a procoagulant effect in vivo, a tail vein transection (TVT) model was employed in HA mice. In this model, one lateral tail vein is transected, and blood is collected over 40 min with clots at the vessel wall disrupted every 10 min as needed. Since GB5 interacts with human and not mouse FV, plasma derived human FV was infused into HA mice. We found that blood loss in the TVT model using HA mice infused with human FV alone (5 μg/mouse) or GB5 alone (20 μg/mouse) was no different than mice infused with PBS (n = 3-5 mice/group except GB5 alone where n = 1: ~600 μl blood loss). In contrast to these data, when human FV and GB5 were co-administered, blood loss was reduced ~3-fold (~200 μl) which was statistically significant relative to the controls but did not reach the level of wild-type mice (blood loss ~50 μl). Overall, the results show that GB5 is a procoagulant monoclonal antibody that binds with high affinity to human FV and influences reactions that lead to its activation and inactivation. We hypothesize that the net effect of GB5 is the enhanced production of FVa which has a major impact on TG. In plasma-based systems with and without the protein C system and an in vivo hemophilic model, GB5 is procoagulant and can reduce bleeding. Our work shows that targeting the common coagulation pathway at the level of FV has benefit in the context of hemophilia and may be a viable and safe therapeutic approach.

Mechanisms Underlining Inflammatory Pain Sensitivity in Mice Selected for High and Low Stress-Induced Analgesia-The Role of Endocannabinoids and Microglia.

In this work we strived to determine whether endocannabinoid system activity could account for the differences in acute inflammatory pain sensitivity in mouse lines selected for high (HA) and low (LA) swim-stress-induced analgesia (SSIA). Mice received intraplantar injections of 5% formalin and the intensity of nocifensive behaviours was scored. To assess the contribution of the endocannabinoid system, mice were intraperitoneally (i.p.) injected with rimonabant (0.3–3 mg/kg) prior to formalin. Minocycline (45 and 100 mg/kg, i.p.) was administered to investigate microglial activation. The possible involvement of the endogenous opioid system was investigated with naloxone (1 mg/kg, i.p.). Cannabinoid receptor types 1 and 2 (Cnr1, Cnr2) and opioid receptor subtype (Oprm1, Oprd1, Oprk1) mRNA levels were quantified by qPCR in the structures of the central nociceptive circuit. Levels of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) were measured by liquid chromatography coupled with the mass spectrometry method (LC-MS/MS). In the interphase, higher pain thresholds in the HA mice correlated with increased spinal anandamide and 2-AG release and higher Cnr1 transcription. Downregulation of Oprd1 and Oprm1 mRNA was noted in HA and LA mice, respectively, however no differences in naloxone sensitivity were observed in either line. As opposed to the LA mice, inflammatory pain sensitivity in the HA mice in the tonic phase was attributed to enhanced microglial activation, as evidenced by enhanced Aif1 and Il-1β mRNA levels. To conclude, Cnr1 inhibitory signaling is one mechanism responsible for decreased pain sensitivity in HA mice in the interphase, while increased microglial activation corresponds to decreased pain thresholds in the tonic inflammatory phase.

Neutrophil Extracellular Traps Promote Joint Injury in Hemophilia

▪Epidemiological evidence suggests that recurring episodes of joint-bleeding contribute to the development of hemophilic arthropathy (H) in 70-85% of hemophilia patients. Despite major advances in the treatment to prevent joint bleeding, HA continues to be a major morbidity affecting hemophilia patients and the etiological mechanism contributing to the progression of HA remains poorly understood. Recent evidence suggests that the accumulation of blood in the joints may lead to the release of erythrocyte-derived DAMPs (eDAMPs) such as heme and hemoglobin that can promote sterile inflammation, however, the innate immune pathways contributing to this pathophysiology remain unknown.In the study, we used a model of puncture-induced knee joint injury in FVIII-total knockout (F8TKO) mice and blood samples from hemophilia-A patients diagnosed with HA. Intravital multi-photon-excitation fluorescence intravital (in vivo) microscopy of injured synovium in live F8TKO or control mice was conducted to assess neutrophil-platelet aggregation and NETs generation in the knee-joint. Imaging-flow-cytometry and ELISA assays were used to estimate the number of circulating NETs in plasma of patients diagnosed with HA and mice after the knee-injury procedure. Scoring of the bleeding severity, histology, IHC and confocal imaging of joints were conducted to quantify the joint injury in mice.F8TKO but not control mice manifested knee-joint injury and severity of bleeding 5-days post knee-injury. Progression of knee-joint injury was associated with increased neutrophil accumulation and NETs shedding within the synovium of F8TKO mice. Circulating NETs were significantly abundant in the plasma of hemophilia patients diagnosed with HA and F8TKO mice following knee-injury but not plasma of control humans or mice.These findings are the first to suggest that NETs contribute to pathogenesis of HA in hemophilia. Currently, experiments are underway to identify the innate immune pathways that promote NETs shedding, leading to joint-damage in hemophilia. DisclosuresRagni: Takeda Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioverativ (Sanofi): Membership on an entity's Board of Directors or advisory committees; BioMarin Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Alnylam (Sanofi): Membership on an entity's Board of Directors or advisory committees; University of Pittsburgh: Research Funding; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees. Sundd: CSL Behring Inc: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding.

Neutrophil Extracellular Traps promote joint damage in hemophilia.

Abstract Hemophilic arthropathy (HA) is the pathophysiology resulting from recurrent joint bleeding in hemophilia. HA leads to joint damage, chronic pain and poor quality of life. Despite advances in the treatment to prevent joint bleeding, HA continues to be a major morbidity affecting hemophilia patients and the mechanism contributing to HA remains elusive. Recent evidence suggests that joint-bleeding may promote the release of erythrocyte and tissue-derived damage-associated-molecular-pattern molecules (DAMPs) that can trigger the sterile inflammation in the joints, however, the innate immune pathways remain unclear. We used a model of knee joint injury-induced HA in FVIII-total knockout (F8TKO) mice and samples from hemophilia patients diagnosed with HA. In vivo multiphoton-excitation fluorescence imaging of injured synovial cavity in live F8TKO or control mice was used to assess NETs formation within joint capsule. Imaging-flow-cytometry and ELISA assays were used to evaluate circulating NETs in HA patients and mice with knee-injury. Bleeding severity scoring, histology, IHC and confocal imaging of joints were conducted to assess the joint injury in mice. F8TKO but not control mice manifested knee-joint injury and severity of bleeding 5-days post knee-injury. Knee-joint injury was associated with increased neutrophil accumulation and NETs shedding within the synovium of F8TKO mice. Circulating NETs were abundant in the plasma of patients diagnosed with HA and F8TKO following injury but not plasma of controls. These findings are the first to suggest that NETs contribute to pathogenesis of HA. Currently, study is underway to identify the innate immune pathways that promote NETs shedding, leading to joint-damage in hemophilia.

Hypertensive Effect of Downregulation of the Opioid System in Mouse Model of Different Activity of the Endogenous Opioid System.

The opioid system is well-known for its role in modulating nociception and addiction development. However, there are premises that the endogenous opioid system may also affect blood pressure. The main goal of the present study was to determine the impact of different endogenous opioid system activity and its pharmacological blockade on blood pressure. Moreover, we examined the vascular function in hyper- and hypoactive states of the opioid system and its pharmacological modification. In our study, we used two mouse lines which are divergently bred for high (HA) and low (LA) swim stress-induced analgesia. The obtained results indicated that individuals with low endogenous opioid system activity have higher basal blood pressure compared to those with a hyperactive opioid system. Additionally, naloxone administration only resulted in the elevation of blood pressure in HA mice. We also showed that the hypoactive opioid system contributes to impaired vascular relaxation independent of endothelium, which corresponded with decreased guanylyl cyclase levels in the aorta. Together, these data suggest that higher basal blood pressure in LA mice is a result of disturbed mechanisms in vascular relaxation in smooth muscle cells. We believe that a novel mechanism which involves endogenous opioid system activity in the regulation of blood pressure will be a promising target for further studies in hypertension development.

Loss of Brain-Derived Neurotrophic Factor (BDNF) Resulting From Congenital- Or Mild Traumatic Brain Injury-Induced Blood–Brain Barrier Disruption Correlates With Depressive-Like Behaviour

Brain-derived neurotrophic factor (BDNF) plays an important role in processes associated with neuroplasticity and neuroprotection. Evidence suggests that decreased BDNF levels in the central nervous system (CNS) represent a mechanism underlying the development of mood disorders. We hypothesize that both congenital and traumatic brain injury (mTBI)-induced blood–brain barrier (BBB) breakdown are responsible for brain BDNF depletion that contributes to the development of depressive-like symptoms. We employed a mouse model of innate differences in BBB integrity with high (HA) and low (LA) permeability. Depressive-like behaviours were determined under chronic mild stress (CMS) conditions or following mTBI using the tail suspension test (TST). Microvascular leakage of the BBB was evaluated using the Evans Blue Dye (EBD) extravasation method. BDNF concentrations in the brain and plasma were measured using the ELISA. Control HA mice with congenitally high BBB permeability showed exacerbated depressive-like behaviours compared with LA mice. In LA mice, with normal BBB function, mTBI, but not CMS, facilitated depressive-like behaviours, which correlated with enhanced BDNF efflux from the brain. In addition, mTBI triggered upregulation of the Bdnf gene in LA mice to compensate for BDNF loss. No alterations in BDNF levels were observed in mTBI and CMS-exposed HA mice. Moreover, CMS did not induce BBB damage or affect depressive-like behaviours in HA mice despite downregulating Bdnf gene expression. To conclude, BDNF efflux through the mTBI-disrupted BBB is strongly linked to the development of depressive-like behaviours, while the depressive phenotype in mice with congenital BBB dysfunction is independent of BDNF leakage.

High-active Mice Have Elevated Clearance Rate Of Bcaas Compared To Low-active Mice

Physical activity (PA) is associated with a diminished prevalence of hypokinetic related diseases and its worldwide economic burden on health care cost. For several decades, branched-chain amino acids (BCAAs; leucine [LEU], isoleucine [ILE], valine [VAL]) have been studied for muscle wasting disorders. However, little is known about the metabolic interactions between BCAAs and regulation of PA levels. PURPOSE: To assess BCAA metabolism by measuring plasma clearance rates in mice previously classified as either low-active (LA; C3H/HeJ) or high-active (HA; C57L/J). METHODS: 12-week-old male LA (n=23) mice (body weight: 25.8 ± 1.2g; lean body mass: 21.0 ± 1.1g; fat mass: 2.5 ± 0.5g) and HA (n=20) mice (27.5 ± 1.2; 22.5 ± 1.3; 2.5 ± 0.7) were used. Under anesthesia, a pulse of stable tracers (L-LEU[13C6], L-ILE [1-13C], and L-VAL [13C5]) was administered via the right jugular vein catheter. Subsequently, blood samples were taken (Time: 1, 3, 5, 7, 10, 15, 20, 25, 30, and 40 mins). Plasma enrichments and concentrations of LEU, ILE, and VAL were determined by LC-MS/MS. Whole-body production (WBP) was calculated from fitted area under the curve (AUC) as pulse/AUC, and clearance of stable tracers was calculated as WBP/plasma concentrations. Fitting and statistical analysis (unpaired student t-tests) were performed using GraphPad Prism 8 software. Data are expressed as mean ± SE. RESULTS: HA mice had significantly lower plasma concentrations for LEU (125.2 ± 4.8 vs 144.8 ± 6.4 μmol/l, p= 0.02), ILE (48.5 ± 1.9 vs 57.7 ± 2.3 μmol/l, p< 0.01) and VAL (156.8 ± 4.9 vs 187.9 ± 7.1 μmol/l, p< 0.01) and significantly higher WBP values for ILE (11.6 ± 0.6 vs 8.3 ± 1.1 nmol/g lbm/m, p= 0.02) and VAL (111.4 ± 5.4 vs 92.8 ± 2.8 nmol/g lbm/m, p< 0.01) compared to LA mice. No significant WBP differences were observed for LEU (75.5 ± 3.3 vs 72.7 ± 3.2 nmol/g lbm/m, p= 0.56). HA mice demonstrated higher percent clearance for LEU (40%), ILE (76%), and VAL (29%) compared to LA mice. CONCLUSIONS: The observed changes in plasma concentration, WBP, and clearance of BCAAs suggest modified metabolic pathways of LEU, VAL, and ILE in HA mice, compared to LA mice. FUNDING SOURCES: Texas A&M Vice President of Research Office, TACSM Student Research Development Award, College of Education & Human Development Student Research Grant, and funds from the Omar Smith Endowment.

Divergent Response to Cannabinoid Receptor Stimulation in High and Low Stress-Induced Analgesia Mouse Lines Is Associated with Differential G-Protein Activation

Bidirectional selection of mice for high (HA) and low (LA) swim stress-induced analgesia (SSIA) is associated with a divergent response to opioids. In the current study, we investigated whether the genetic divergence in opioid system activity between HA and LA mice also affects cannabinoid sensitivity. Additionally, we also investigated whether the endocannabinoid system mediates SSIA in these lines. Numerous reports support the existence of pharmacological and molecular interactions between the opioid and cannabinoid systems along the pain pathways, as both systems utilize the same G-protein subtype for signal transduction. Mice from both lines were treated with a non-selective CB1/CB2 agonist, WIN55,212–2 and their behavior was evaluated according to the tetrad paradigm assessing antinociception, catalepsy, hypothermia and locomotor activity. Surprisingly, the engagement of CB1 receptors in SSIA was not confirmed. G-protein activation was studied in different brain regions and the spinal cord in the [35S]GTPγS assay. It was shown that WIN55,212–2 produced more potent antinociception in HA than in LA mice. Also, HA mice displayed stronger cannabinoid-induced catalepsy in the bar test. However, LA mice were more sensitive to the hypothermic effect of WIN55,212–2. The intensity of behavioral responses to WIN55,212–2 was correlated with increased G-protein activation in the periaqueductal gray matter, frontal cortex, striatum and thalamus in HA mice. A weak response to WIN55,212–2 in LA mice could depend on impaired CB2 receptor signaling. In conclusion, differences in both opioid and cannabinoid sensitivity between HA and LA mice could stem from alterations in intracellular second messenger mechanisms involving G-protein activation.

Potential long-term treatment of hemophilia A by neonatal co-transplantation of cord blood-derived endothelial colony-forming cells and placental mesenchymal stromal cells

BackgroundHemophilia A (HA) is an X-linked recessive disorder caused by mutations in the Factor VIII (FVIII) gene leading to deficient blood coagulation. As a monogenic disorder, HA is an ideal target for cell-based gene therapy, but successful treatment has been hampered by insufficient engraftment of potential therapeutic cells.MethodsIn this study, we sought to determine whether co-transplantation of endothelial colony-forming cells (ECFCs) and placenta-derived mesenchymal stromal cells (PMSCs) can achieve long-term engraftment and FVIII expression. ECFCs and PMSCs were transduced with a B domain deleted factor VIII (BDD-FVIII) expressing lentiviral vector and luciferase, green fluorescent protein or Td-Tomato containing lentiviral tracking vectors. They were transplanted intramuscularly into neonatal or adult immunodeficient mice.ResultsIn vivo bioluminescence imaging showed that the ECFC only and the co-transplantation groups but not the PMSCs only group achieved long-term engraftment for at least 26 weeks, and the co-transplantation group showed a higher engraftment than the ECFC only group at 16 and 20 weeks post-transplantation. In addition, cell transplantation at the neonatal age achieved higher engraftment than at the adult age. Immunohistochemical analyses further showed that the engrafted ECFCs expressed FVIII, maintained endothelial phenotype, and generated functional vasculature. Next, co-transplantation of ECFCs and PMSCs into F8 knock-out HA mice reduced the blood loss volume from 562.13 ± 19.84 μl to 155.78 ± 44.93 μl in a tail-clip assay.ConclusionsThis work demonstrated that co-transplantation of ECFCs with PMSCs at the neonatal age is a potential strategy to achieve stable, long-term engraftment, and thus holds great promise for cell-based treatment of HA.

Ethanol consumption following mild traumatic brain injury is related to blood-brain barrier permeability.

Several preclinical and clinical studies that deal with the neuropathological consequences of mild traumatic brain injury (mTBI) have focused on unraveling its effect on ethanol drinking behavior. Previous reports describe changes in ethanol consumption, both in animal models of mTBI as well as in patients, after concussive brain injury. However, the neurobiological mechanisms underlying this phenomenon are still poorly understood. In the present study, we used a unique model of mouse lines divergently selected for high (HA) or low (LA) swim stress-induced analgesia to examine the effect of mTBI on ethanol drinking behavior. In comparison with LA mice, their HA counterparts exhibited increased blood-brain barrier (BBB) permeability, lower basal alcohol preference, and lower level of stress-induced ethanol intake. Here, we showed that mTBI attenuates voluntary ethanol intake in LA, but not in HA mice. Interestingly, BBB disruption after mannitol infusion also decreases the level of ethanol drinking behavior in this line. We conclude that in alcohol-preferring LA mice, BBB disruption as a consequence of mTBI attenuates ethanol consumption. Our results suggest that the innate level of BBB integrity plays a pivotal role in regulation of ethanol consumption in mice showing differential endogenous opioid system activity.

Bidirectional selection for high and low stress-induced analgesia affects G-protein activity

Mice selected for high (HA) and low (LA) swim stress-induced analgesia (SSIA) are a unique model for studying the genetic background of this phenomenon. HA and LA miceshow substantial differences in the magnitude of the antinociceptive response to stress and when treated with exogenous opioids. However, the direct cause underplaying this distinctive feature has not yet been identified. The current study was designed to investigate the possibility that disturbances in G-protein signaling could explain the divergent response to opioid agonists. Supraspinal and spinal opioid sensitivity was assessed in vivo with intraperitoneal morphine and subsequent thermal stimulus exposure. The level of opioid receptor-mediated G-protein activation was investigated by means of DAMGO and morphine-stimulated [35S]GTPγS assay in the brain and spinal cord homogenates from HA and LA mice. Morphine (3–249 μmol/kg, i.p) was over 6 - and 3 - times more potent in HA than LA mice in the hot plate and tail-flick assays, respectively. Additionally, HA mice showed elevated β - endorphin levels in the brain. Enhanced efficacy of agonist-stimulated [35S]GTPγS binding was detected in opioid receptor-rich limbic regions of HA mice like the hypothalamus and hippocampus. Increased G-protein activity also emerged in the thalamus, periaqueductal gray matter and prefrontal cortex. In conclusion, the magnitude of the antinociceptive response to opioids in HA and LA mice is correlated with alterations in G-protein activation in brain regions responsible for integration and descending modulation of nociceptive information as well as at sites governing the emotional response to stressful stimuli.

Defective TAFI activation in hemophilia A mice is a major contributor to joint bleeding

AbstractJoint bleeds are common in congenital hemophilia but rare in acquired hemophilia A (aHA) for reasons unknown. To identify key mechanisms responsible for joint-specific bleeding in congenital hemophilia, bleeding phenotypes after joint injury and tail transection were compared in aHA wild-type (WT) mice (receiving an anti–factor VIII [FVIII] antibody) and congenital HA (FVIII−/−) mice. Both aHA and FVIII−/− mice bled severely after tail transection, but consistent with clinical findings, joint bleeding was notably milder in aHA compared with FVIII−/− mice. Focus was directed to thrombin-activatable fibrinolysis inhibitor (TAFI) to determine its potentially protective effect on joint bleeding in aHA. Joint bleeding in TAFI−/− mice with anti-FVIII antibody was increased, compared with WT aHA mice, and became indistinguishable from joint bleeding in FVIII−/− mice. Measurements of circulating TAFI zymogen consumption after joint injury indicated severely defective TAFI activation in FVIII−/− mice in vivo, consistent with previous in vitro analyses in FVIII-deficient plasma. In contrast, notable TAFI activation was observed in aHA mice, suggesting that TAFI protected aHA joints against bleeding. Pharmacological inhibitors of fibrinolysis revealed that urokinase-type plasminogen activator (uPA)–induced fibrinolysis drove joint bleeding, whereas tissue-type plasminogen activator–mediated fibrinolysis contributed to tail bleeding. These data identify TAFI as an important modifier of hemophilic joint bleeding in aHA by inhibiting uPA-mediated fibrinolysis. Moreover, our data suggest that bleed protection by TAFI was absent in congenital FVIII−/− mice because of severely defective TAFI activation, underscoring the importance of clot protection in addition to clot formation when considering prohemostatic strategies for hemophilic joint bleeding.

Altered serotonergic and GABAergic neurotransmission in a mice model of obsessive-compulsive disorder

There is ample evidence that obsessive-compulsive disorder (OCD) is based on reduced serotonergic function. Replicated bidirectional selection for thermoregulatory nest-building behavior in the laboratory house mouse (Mus musculus) resulted in compulsive-like, non-compulsive-like and randomly bred control mice that represent a non-induced animal model of OCD. The present study aimed at investigating the neurochemical patterns in specific brain regions of compulsive-like (HA) versus non-compulsive-like (LA) and normal (CA) mice. The neurochemical investigation of several brain regions of the corticostriato-thalamocortical circuity, i.e., nucleus caudatus (CPU), nucleus accumbens (NAc), globus pallidus (GP), hippocampus (HPC), amygdala (AM), ventral tegmental area (VTA) and substantia nigra (SN) was performed by electrochemical (serotonin and dopamine) and fluorescence (glutamate and GABA) HPLC detection. HA mice displayed significantly decreased 5-HT concentrations in the mPFC and LA mice displayed a significant increase in GABA concentrations in the mPFC. This supports the pathophysiological relevance of serotonin in the manifestation of OCD and adding to the construct validity of the non-induced mouse model of OCD.

Delta-opioid receptor antagonism leads to excessive ethanol consumption in mice with enhanced activity of the endogenous opioid system

The opioid system modulates the central reinforcing effects of ethanol and participates in the etiology of addiction. However, the pharmacotherapy of ethanol dependence targeted on the opioid system is little effective and varies due to individual patients' sensitivity. In the present study, we used two mouse lines with high (HA) and low (LA) activity of the endogenous opioid system to analyze the effect of opioid receptor blockade on ethanol drinking behavior. We found that LA and HA lines characterized by divergent magnitudes of swim stress-induced analgesia also differ in ethanol intake and preference. Downregulation of the opioid system in LA mice was associated with increased ethanol consumption. Treatment with a non-selective opioid receptor antagonist (naloxone) had no effect on ethanol intake in this line. Surprisingly, in HA mice, the blockage of opioid receptors led to excessive ethanol consumption. Moreover, naloxone selectively induced high levels of anxiety- and depressive-like behaviors in HA mice which was attenuated by ethanol. With the use of specific opioid receptor antagonists we showed that the naloxone-induced increase in ethanol drinking in HA mice is mediated mainly by δ and to a lower extent by μ opioid receptors. The effect of δ-opioid receptor antagonism was abolished in HA mice carrying a C320T transition in the δ-opioid receptor gene (EU446125.1), which impairs this receptor's function. Our results indicate that high activity of the opioid system plays a protective role against ethanol dependence. Therefore, its blockage with opioid receptor antagonists may lead to a profound increase in ethanol consumption.

Naloxone exacerbates memory impairments and depressive-like behavior after mild traumatic brain injury (mTBI) in mice with upregulated opioid system activity

The neuroprotective role of the endogenous opioid system in the pathophysiological sequelae of brain injury remains largely ambiguous. Noteworthy, almost no data is available on how its genetically determined activity influences the outcome of mild traumatic brain injury. Thus, the aim of our study was to examine the effect of opioid receptor blockage on cognitive impairments produced by mild traumatic brain injury in mice selectively bred for high (HA) and low (LA) swim-stress induced analgesia that show innate divergence in opioid system activity. Mild traumatic brain injury was induced with a weight-drop device on anaesthetized mice. Naloxone (5mg/kg) was intraperitoneally delivered twice a day for 7days to non-selectively block opioid receptors. Spatial memory performance and manifestations of depressive-like behavior were assessed using the Morris Water Maze and tail suspension tests, respectively. Mild traumatic brain injury resulted in a significant deterioration of spatial memory performance and severity of depressive-like behavior in the LA mouse line as opposed to HA mice. Opioid receptor blockage with naloxone unmasked cognitive deficits in HA mice but was without effect in the LA line. The results suggest a protective role of genetically predetermined enhanced opioid system activity in suppression of mild brain trauma-induced cognitive impairments. Mice selected for high and low swim stress-induced analgesia might therefore be a useful model to study the involvement of the opioid system in the pathophysiology and neurological outcome of traumatic brain injury.