Heart failure-inducible gene therapy targeting protein phosphatase 1 prevents progressive left ventricular remodeling

Background

The targeting of Ca²⁺ cycling has emerged as a potential therapy for the treatment of severe heart failure. These approaches include gene therapy directed at overexpressing sarcoplasmic reticulum (SR) Ca²⁺ ATPase, or ablation of phospholamban (PLN) and associated protein phosphatase 1 (PP1) protein complexes. We previously reported that PP1β, one of the PP1 catalytic subunits, predominantly suppresses Ca²⁺ uptake in the SR among the three PP1 isoforms, thereby contributing to Ca²⁺ downregulation in failing hearts. In the present study, we investigated whether heart-failure-inducible PP1β-inhibition by adeno-associated viral-9 (AAV9) vector mediated gene therapy is beneficial for preventing disease progression in genetic cardiomyopathic mice.

Methods

We created an adeno-associated virus 9 (AAV9) vector encoding PP1β short-hairpin RNA (shRNA) or negative control (NC) shRNA. A heart failure inducible gene expression system was employed using the B-type natriuretic protein (BNP) promoter conjugated to emerald-green fluorescence protein (EmGFP) and the shRNA sequence. AAV9 vectors (AAV9-BNP-EmGFP-PP1βshRNA and AAV9-BNP-EmGFP-NCshRNA) were injected into the tail vein (2×10¹¹ GC/mouse) of muscle LIM protein deficient mice (MLPKO), followed by serial analysis of echocardiography, hemodynamic measurement, biochemical and histological analysis at 3 months.

Results

In the MLPKO mice, BNP promoter activity was shown to be increased by detecting both EmGFP expression and the induced reduction of PP1β by 25% in the myocardium. Inducible PP1βshRNA delivery preferentially ameliorated left ventricular diastolic function and mitigated adverse ventricular remodeling. PLN phosphorylation was significantly augmented in the AAV9-BNP-EmGFP-PP1βshRNA injected hearts compared with the AAV9-BNP-EmGFP-NCshRNA group. Furthermore, BNP production was reduced, and cardiac interstitial fibrosis was abrogated at 3 months.

Conclusion

Heart failure-inducible molecular targeting of PP1β has potential as a novel therapeutic strategy for heart failure.     

Interleukin-1β and Interleukin-1 Receptor Antagonist Appear in Grey Matter Additionally to White Matter Lesions during Experimental Multiple Sclerosis

Background

Multiple sclerosis (MS) has been mainly attributed to white matter (WM) pathology. However, recent evidence indicated the presence of grey matter (GM) lesions. One of the principal mediators of inflammatory processes is interleukin-1β (IL-1β), which is known to play a role in MS pathogenesis. It is unknown whether IL-1β is solely present in WM or also in GM lesions. Using an experimental MS model, we questioned whether IL-1β and the IL-1 receptor antagonist (IL-1ra) are present in GM in addition to affected WM regions.

Methods

The expression of IL-1β and IL-1ra in chronic-relapsing EAE (cr-EAE) rats was examined using in situ hybridization, immunohistochemistry and real-time PCR. Rats were sacrificed at the peak of the first disease phase, the trough of the remission phase, and at the peak of the relapse. Histopathological characteristics of CNS lesions were studied using immunohistochemistry for PLP, CD68 and CD3 and Oil-Red O histochemistry.

Results

IL-1β and IL-ra expression appears to a similar extent in affected GM and WM regions in the brain and spinal cord of cr-EAE rats, particularly in perivascular and periventricular locations. IL-1β and IL-1ra expression was dedicated to macrophages and/or activated microglial cells, at sites of starting demyelination. The time-dependent expression of IL-1β and IL-1ra revealed that within the spinal cord IL-1β and IL-1ra mRNA remained present throughout the disease, whereas in the brain their expression disappeared during the relapse.

Conclusions

The appearance of IL-1β expressing cells in GM within the CNS during cr-EAE may explain the occurrence of several clinical deficits present in EAE and MS which cannot be attributed solely to the presence of IL-1β in WM. Endogenously produced IL-1ra seems not capable to counteract IL-1β-induced effects. We put forward that IL-1β may behold promise as a target to address GM, in addition to WM, related pathology in MS.     

Colocalization of 14-3-3 proteins with SOD1 in Lewy body-like hyaline inclusions in familial amyotrophic lateral sclerosis cases and the animal model

Background and Purpose

Cu/Zn superoxide dismutase (SOD1) is a major component of Lewy body-like hyaline inclusion (LBHI) found in the postmortem tissue of SOD1-linked familial amyotrophic lateral sclerosis (FALS) patients. In our recent studies, 14-3-3 proteins have been found in the ubiquitinated inclusions inside the anterior horn cells of spinal cords with sporadic amyotrophic lateral sclerosis (ALS). To further investigate the role of 14-3-3 proteins in ALS, we performed immunohistochemical analysis of 14-3-3 proteins and compared their distributions with those of SOD1 in FALS patients and SOD1-overexpressing mice.

Methods

We examined the postmortem brains and the spinal cords of three FALS cases (A4V SOD1 mutant). Transgenic mice expressing the G93A mutant human SOD1 (mutant SOD1-Tg mice), transgenic mice expressing the wild-type human SOD1 (wild-type SOD1-Tg mice), and non-Tg wild-type mice were also subjected to the immunohistochemical analysis.

Results

In all the FALS patients, LBHIs were observed in the cytoplasm of the anterior horn cells, and these inclusions were immunopositive intensely for pan 14-3-3, 14-3-3β, and 14-3-3γ. In the mutant SOD1-Tg mice, a high degree of immunoreactivity for misfolded SOD1 (C4F6) was observed in the cytoplasm, with an even greater degree of immunoreactivity present in the cytoplasmic aggregates of the anterior horn cells in the lumbar spinal cord. Furthermore, we have found increased 14-3-3β and 14-3-3γ immunoreactivities in the mutant SOD1-Tg mice. Double immunofluorescent staining showed that C4F6 and 14-3-3 proteins were partially co-localized in the spinal cord with FALS and the mutant SOD1-Tg mice. In comparison, the wild-type SOD1-Tg and non-Tg wild-type mice showed no or faint immunoreactivity for C4F6 and 14-3-3 proteins (pan 14-3-3, 14-3-3β, and 14-3-3γ) in any neuronal compartments.

Discussion

These results suggest that 14-3-3 proteins may be associated with the formation of SOD1-containing inclusions, in FALS patients and the mutant SOD1-Tg mice.     

AAV-Mediated Gene Delivery in a Feline Model of Sandhoff Disease Corrects Lysosomal Storage in the Central Nervous System

Sandhoff disease (SD) is an autosomal recessive neurodegenerative disease caused by a mutation in the gene for the β-subunit of β-N-acetylhexosaminidase (Hex), resulting in the inability to catabolize ganglioside GM2 within the lysosomes. SD presents with an accumulation of GM2 and its asialo derivative GA2, primarily in the central nervous system. Myelin-enriched glycolipids, cerebrosides and sulfatides, are also decreased in SD corresponding with dysmyelination. At present, no treatment exists for SD. Previous studies have shown the therapeutic benefit of adeno-associated virus (AAV) vector-mediated gene therapy in the treatment of SD in murine and feline models. In this study, we treated presymptomatic SD cats with AAVrh8 vectors expressing feline Hex in the thalamus combined with intracerebroventricular (Thal/ICV) injections. Treated animals showed clearly improved neurologic function and quality of life, manifested in part by prevention or attenuation of whole-body tremors characteristic of untreated animals. Hex activity was significantly elevated, whereas storage of GM2 and GA2 was significantly decreased in tissue samples taken from the cortex, cerebellum, thalamus, and cervical spinal cord. Treatment also increased levels of myelin-enriched cerebrosides and sulfatides in the cortex and thalamus. This study demonstrates the therapeutic potential of AAV for feline SD and suggests a similar potential for human SD patients.     

Functional and Behavioral Restoration of Vision by Gene Therapy in the Guanylate Cyclase-1 (GC1) Knockout Mouse

Background

Recessive mutations in guanylate cyclase-1 (Gucy2d) are associated with severe, early onset Leber congenital amaurosis-1(LCA1). Gucy2d encodes guanylate cyclase (GC1) is expressed in photoreceptor outer segment membranes and produces cGMP in these cells. LCA1 patients present in infancy with severely impaired vision and extinguished electroretinogram (ERG) but retain some photoreceptors in both their macular and peripheral retina for years. Like LCA1 patients, loss of cone function in the GC1 knockout (GC1KO) mouse precedes cone degeneration. The purpose of this study was to test whether delivery of functional GC1 to cone cells of the postnatal GC1KO mouse could restore function to these cells.

Methodology/Principal Findings

Serotype 5 AAV vectors containing either a photoreceptor-specific, rhodopsin kinase (hGRK1) or ubiquitous (smCBA) promoter driving expression of wild type murine GC1 were subretinally delivered to one eye of P14 GC1KO mice. Visual function (ERG) was analyzed in treated and untreated eyes until 3 months post injection. AAV-treated, isogenic wild type and uninjected control mice were evaluated for restoration of visual behavior using optomotor testing. At 3 months post injection, all animals were sacrificed, and their treated and untreated retinas assayed for expression of GC1 and localization of cone arrestin. Cone-mediated function was restored to treated eyes of GC1KO mice (ERG amplitudes were ∼45% of normal). Treatment effect was stable for at least 3 months. Robust improvements in cone-mediated visual behavior were also observed, with responses of treated mice being similar or identical to that of wild type mice. AAV-vectored GC1 expression was found in photoreceptors and cone cells were preserved in treated retinas.

Conclusions/Significance

This is the first demonstration of gene-based restoration of both visual function/vision-elicited behavior and cone preservation in a mammalian model of GC1 deficiency. Importantly, results were obtained using a well characterized, clinically relevant AAV vector. These results lay the ground work for the development of an AAV-based gene therapy vector for the treatment of LCA1.     

Human neural stem cell replacement therapy for amyotrophic lateral sclerosis by spinal transplantation

Background

Mutation in the ubiquitously expressed cytoplasmic superoxide dismutase (SOD1) causes an inherited form of Amyotrophic Lateral Sclerosis (ALS). Mutant synthesis in motor neurons drives disease onset and early disease progression. Previous experimental studies have shown that spinal grafting of human fetal spinal neural stem cells (hNSCs) into the lumbar spinal cord of SOD1^G93A rats leads to a moderate therapeutical effect as evidenced by local α-motoneuron sparing and extension of lifespan. The aim of the present study was to analyze the degree of therapeutical effect of hNSCs once grafted into the lumbar spinal ventral horn in presymptomatic immunosuppressed SOD1^G93A rats and to assess the presence and functional integrity of the descending motor system in symptomatic SOD1^G93A animals.

Methods/Principal Findings

Presymptomatic SOD1^G93A rats (60–65 days old) received spinal lumbar injections of hNSCs. After cell grafting, disease onset, disease progression and lifespan were analyzed. In separate symptomatic SOD1^G93A rats, the presence and functional conductivity of descending motor tracts (corticospinal and rubrospinal) was analyzed by spinal surface recording electrodes after electrical stimulation of the motor cortex. Silver impregnation of lumbar spinal cord sections and descending motor axon counting in plastic spinal cord sections were used to validate morphologically the integrity of descending motor tracts. Grafting of hNSCs into the lumbar spinal cord of SOD1^G93A rats protected α-motoneurons in the vicinity of grafted cells, provided transient functional improvement, but offered no protection to α-motoneuron pools distant from grafted lumbar segments. Analysis of motor-evoked potentials recorded from the thoracic spinal cord of symptomatic SOD1^G93A rats showed a near complete loss of descending motor tract conduction, corresponding to a significant (50–65%) loss of large caliber descending motor axons.

Conclusions/Significance

These data demonstrate that in order to achieve a more clinically-adequate treatment, cell-replacement/gene therapy strategies will likely require both spinal and supraspinal targets.     

Prostaglandin E2 reverses aberrant production of an inflammatory chemokine by microglia from Sandhoff disease model mice through the cAMP-PKA pathway

Background

Sandhoff disease (SD) is a neurodegenerative lysosomal β-hexosaminidase (Hex) deficiency involving excessive accumulation of undegraded substrates, including terminal GlcNAc-oligosaccharides and GM2 ganglioside. Microglia-mediated neuroinflammation contributes to the pathogenesis and progression of SD. Our previous study demonstrated that MIP-1α, a putative pathogenic factor for SD, is up-regulated in microglial cells derived from SD model mice (SD-Mg) through activation of Akt and JNK.

Methodology/Principal Findings

In this study, we first demonstrated that prostaglandin E2 (PGE2), which is one of the lipid mediators derived from arachidonic acid and is known to suppress activation of microglia, reduced the aberrant MIP-1α production by SD-Mg to the same level as by WT-Mg. PGE2 also attenuated the activation of Akt and JNK. The inhibition of MIP-1α production and the activation of Akt and JNK occurred through the EP2 and 4/cAMP/PKA signaling pathway in the murine microglia derived from SD model mice.

Conclusions/Significance

We propose that PGE2 plays a role as a negative regulator of MIP-1α production in the pathogenesis of SD, and that PGE2-EP2 and 4/cAMP/PKA signaling could be a target pathway for therapy for SD.     

General anesthetics inhibit erythropoietin induction under hypoxic conditions in the mouse brain

Background

Erythropoietin (EPO), originally identified as a hematopoietic growth factor produced in the kidney and fetal liver, is also endogenously expressed in the central nervous system (CNS). EPO in the CNS, mainly produced in astrocytes, is induced under hypoxic conditions in a hypoxia-inducible factor (HIF)-dependent manner and plays a dominant role in neuroprotection and neurogenesis. We investigated the effect of general anesthetics on EPO expression in the mouse brain and primary cultured astrocytes.

Methodology/Principal Findings

BALB/c mice were exposed to 10% oxygen with isoflurane at various concentrations (0.10–1.0%). Expression of EPO mRNA in the brain was studied, and the effects of sevoflurane, halothane, nitrous oxide, pentobarbital, ketamine, and propofol were investigated. In addition, expression of HIF-2α protein was studied by immunoblotting. Hypoxia-induced EPO mRNA expression in the brain was significantly suppressed by isoflurane in a concentration-dependent manner. A similar effect was confirmed for all other general anesthetics. Hypoxia-inducible expression of HIF-2α protein was also significantly suppressed with isoflurane. In the experiments using primary cultured astrocytes, isoflurane, pentobarbital, and ketamine suppressed hypoxia-inducible expression of HIF-2α protein and EPO mRNA.

Conclusions/Significance

Taken together, our results indicate that general anesthetics suppress activation of HIF-2 and inhibit hypoxia-induced EPO upregulation in the mouse brain through a direct effect on astrocytes.     

Molecular Mechanisms Underlying the Analgesic Property of Intrathecal Dexmedetomidine and Its Neurotoxicity Evaluation: An In Vivo and In Vitro Experimental Study

Background

Dexmedetomidine (DEX) has been used under perioperative settings as an adjuvant to enhance the analgesic property of local anesthetics by some anesthesiologists. However, the analgesic mechanisms and neurotoxicity of DEX were poorly understood. This study examined the effect of DEX alone on inflammatory pain, and it also examined the underlying molecular mechanisms of DEX in the spinal cord. Furthermore, in vivo and in vitro experiments were performed to investigate the neurotoxicity of DEX on the spinal cord and cortical neurons.

Methods

This study used adult, male Kunming mice. In the acute inflammatory model, the left hind-paws of mice were intradermally injected with pH 5.0 PBS while chronic constrictive injury (CCI) of the sciatic nerve was used to duplicate the neuropathic pain condition. Thermal paw withdrawal latency and mechanical paw withdrawal threshold were tested with a radiant heat test and the Von Frey method, respectively. Locomotor activity and motor coordination were evaluated using the inverted mesh test. Western blotting examined spinal ERK1/2, p-ERK1/2, caspase-3 and β-actin expressions, while spinal c-Fos protein expression was realized with immunohistochemical staining. Hematoxylin eosin (HE) staining was used to examine the pathological impacts of intrathecal DEX on the spinal cord. DAPI (4′,6-diamidino-2-phenylindole) staining was used to observe cell death under an immunofluorescence microscope.

Results

Intra-plantar pH 5.0 PBS-induced acute pain required spinal ERK1/2 activation. Inhibition of spinal ERK1/2 signaling by intrathecal injection of DEX displayed a robust analgesia, via a α2-receptor dependent manner. The analgesic properties of DEX were validated in CCI mice. In vivo studies showed that intrathecal DEX has no significant pathological impacts on the spinal cord, and in vitro experiments indicated that DEX has potential protective effects of lidocaine-induced neural cell death.

Conclusion

Intrathecal injection of DEX alone or as an adjuvant might be potential for pain relief.     

Gene therapy in a humanized mouse model of familial hypercholesterolemia leads to marked regression of atherosclerosis

Background

Familial hypercholesterolemia (FH) is an autosomal codominant disorder caused by mutations in the low-density lipoprotein receptor (LDLR) gene. Homozygous FH patients (hoFH) have severe hypercholesterolemia leading to life threatening atherosclerosis in childhood and adolescence. Mice with germ line interruptions in the Ldlr and Apobec1 genes (Ldlr^−/−Apobec1^−/−) simulate metabolic and clinical aspects of hoFH, including atherogenesis on a chow diet.

Methods/Principal Findings

In this study, vectors based on adeno-associated virus 8 (AAV8) were used to deliver the gene for mouse Ldlr (mLDLR) to the livers of Ldlr^−/−Apobec1^−/− mice. A single intravenous injection of AAV8.mLDLR was found to significantly reduce plasma cholesterol and non-HDL cholesterol levels in chow-fed animals at doses as low as 3×10⁹ genome copies/mouse. Whereas Ldlr^−/−Apobec1^−/− mice fed a western-type diet and injected with a control AAV8.null vector experienced a further 65% progression in atherosclerosis over 2 months compared with baseline mice, Ldlr^−/−Apobec1^−/− mice treated with AAV8.mLDLR realized an 87% regression of atherosclerotic lesions after 3 months compared to baseline mice. Immunohistochemical analyses revealed a substantial remodeling of atherosclerotic lesions.

Conclusions/Significance

Collectively, the results presented herein suggest that AAV8-based gene therapy for FH may be feasible and support further development of this approach. The pre-clinical data from these studies will enable for the effective translation of gene therapy into the clinic for treatment of FH.     

IL-1 and IL-23 mediate early IL-17A production in pulmonary inflammation leading to late fibrosis

Background

Idiopathic pulmonary fibrosis is a devastating as yet untreatable disease. We demonstrated recently the predominant role of the NLRP3 inflammasome activation and IL-1β expression in the establishment of pulmonary inflammation and fibrosis in mice.

Methods

The contribution of IL-23 or IL-17 in pulmonary inflammation and fibrosis was assessed using the bleomycin model in deficient mice.

Results

We show that bleomycin or IL-1β-induced lung injury leads to increased expression of early IL-23p19, and IL-17A or IL-17F expression. Early IL-23p19 and IL-17A, but not IL-17F, and IL-17RA signaling are required for inflammatory response to BLM as shown with gene deficient mice or mice treated with neutralizing antibodies. Using FACS analysis, we show a very early IL-17A and IL-17F expression by RORγt⁺ γδ T cells and to a lesser extent by CD4αβ⁺ T cells, but not by iNKT cells, 24 hrs after BLM administration. Moreover, IL-23p19 and IL-17A expressions or IL-17RA signaling are necessary to pulmonary TGF-β1 production, collagen deposition and evolution to fibrosis.

Conclusions

Our findings demonstrate the existence of an early IL-1β-IL-23-IL-17A axis leading to pulmonary inflammation and fibrosis and identify innate IL-23 and IL-17A as interesting drug targets for IL-1β driven lung pathology.     

The effect of hepatic encephalopathy, hepatic failure, and portosystemic shunt on brain volume of cirrhotic patients: a voxel-based morphometry study

Purpose

To evaluate the effect of hepatic encephalopathy (HE), hepatic failure, and portosystemic shunt (PS) on the brain volume alteration in cirrhotic patients with MRI voxel-based morphometry (VBM).

Methods

Sixty cirrhotic patients (overt HE [OHE], n = 11; minimal HE [MHE], n = 19; non HE [nHE], n = 30) including 12 with pre- and post-transjugular intrahepatic portosystemic shunt (TIPS) scanning and 40 healthy controls were recruited. Neuropsychological and laboratory tests were performed in all patients. VBM was analyzed with ANOVA test among 4 groups, and t-tests for patients with different hepatic function, PS scores, and TIPS. Multiple linear regression was performed to investigate the effect of venous blood ammonia levels, Child-Pugh scores, and PS on the brain volumes in all patients.

Results

Cirrhotic patients exhibited decreased volume in many areas of gray matter (GM), increased volume in thalamus, and increased whiter matter (WM) volume, with the extent of affected brain volume greater in HE patients than nHE patients. Hepatic failure also resulted in decreased GM volume. Patients with high PS scores and TIPS displayed decreased GM and increased WM volume in some regions. Post-TIPS patients displayed increased GM volume in the thalamus. Multiple covariate regression results suggested that Child-Pugh score was a major factor to affect GM volume, while PS mainly affected WM volume.

Conclusion

Brain structure abnormalities appeared bilaterally symmetrical in cirrhotic patients, and the impairment was more extensive in HE patients than those without HE. Increased thalamus volume was not associated with HE progression. Hepatic failure and PS altered cirrhotic patients’ brain structure.     

Central Administration of C-X-C Chemokine Receptor Type 4 Antagonist Alleviates the Development and Maintenance of Peripheral Neuropathic Pain in Mice

Aim

To explore the roles of C-X-C chemokine receptor type 4 (CXCR4) in spinal processing of neuropathic pain at the central nervous system (CNS).

Methods

Peripheral neuropathic pain (PNP) induced by partial sciatic nerve ligation (pSNL) model was assessed in mice. Effects of a single intrathecal (central) administration of AMD3100 (intrathecal AMD3100), a CXCR4 antagonist, on pain behavior and pain-related spinal pathways and molecules in the L3-L5 spinal cord segment was studied compare to saline treatment.

Results

Rotarod test showed that intrathecal AMD3100 did not impair mice motor function. In pSNL-induced mice, intrathecal AMD3100 delayed the development of mechanical allodynia and reversed the established mechanical allodynia in a dose-dependent way. Moreover, intrathecal AMD3100 downregulated the activation of JNK1 and p38 pathways and the protein expression of p65 as assessed by western blotting. Real-time PCR test also demonstrated that substance P mRNA was decreased, while adrenomedullin and intercellular adhesion molecule mRNA was increased following AMD3100 treatment.

Conclusion

Our results suggest that central (spinal) CXCR4 is involved in the development and maintenance of PNP and the regulation of multiple spinal molecular events under pain condition, implicating that CXCR4 would potentially be a therapeutic target for chronic neuropathic pain.     

AMPK regulates circadian rhythms in a tissue- and isoform-specific manner

Background

AMP protein kinase (AMPK) plays an important role in food intake and energy metabolism, which are synchronized to the light-dark cycle. In vitro, AMPK affects the circadian rhythm by regulating at least two clock components, CKIα and CRY1, via direct phosphorylation. However, it is not known whether the catalytic activity of AMPK actually regulates circadian rhythm in vivo.

Methodology/Principal Finding

The catalytic subunit of AMPK has two isoforms: α1 and α2. We investigate the circadian rhythm of behavior, physiology and gene expression in AMPKα1−/− and AMPKα2−/− mice. We found that both α1−/− and α2−/− mice are able to maintain a circadian rhythm of activity in dark-dark (DD) cycle, but α1−/− mice have a shorter circadian period whereas α2−/− mice showed a tendency toward a slightly longer circadian period. Furthermore, the circadian rhythm of body temperature was dampened in α1−/− mice, but not in α2−/− mice. The circadian pattern of core clock gene expression was severely disrupted in fat in α1−/− mice, but it was severely disrupted in the heart and skeletal muscle of α2−/− mice. Interestingly, other genes that showed circadian pattern of expression were dysreguated in both α1−/− and α2−/− mice. The circadian rhythm of nicotinamide phosphoryl-transferase (NAMPT) activity, which converts nicotinamide (NAM) to NAD⁺, is an important regulator of the circadian clock. We found that the NAMPT rhythm was absent in AMPK-deficient tissues and cells.

Conclusion/Significance

This study demonstrates that the catalytic activity of AMPK regulates circadian rhythm of behavior, energy metabolism and gene expression in isoform- and tissue-specific manners.     

The spinal cord expression of neuronal and inducible nitric oxide synthases and their contribution in the maintenance of neuropathic pain in mice

Background

Nitric oxide generated by neuronal (NOS1), inducible (NOS2) or endothelial (NOS3) nitric oxide synthases contributes to pain processing, but the exact role of NOS1 and NOS2 in the maintenance of chronic peripheral neuropathic pain as well as the possible compensatory changes in their expression in the spinal cord of wild type (WT) and NOS knockout (KO) mice at 21 days after total sciatic nerve ligation remains unknown.

Methodology/Principal Findings

The mechanical and thermal allodynia as well as thermal hyperalgesia induced by sciatic nerve injury was evaluated in WT, NOS1-KO and NOS2-KO mice from 1 to 21 days after surgery. The mRNA and protein levels of NOS1, NOS2 and NOS3 in the spinal cord of WT and KO mice, at 21 days after surgery, were also assessed. Sciatic nerve injury led to a neuropathic syndrome in WT mice, in contrast to the abolished mechanical allodynia and thermal hyperalgesia as well as the decreased or suppressed thermal allodynia observed in NOS1-KO and NOS2-KO animals, respectively. Sciatic nerve injury also increases the spinal cord expression of NOS1 and NOS2 isoforms, but not of NOS3, in WT and NOS1-KO mice respectively. Moreover, the presence of NOS2 is required to increase the spinal cord expression of NOS1 whereas an increased NOS1 expression might avoid the up-regulation of NOS2 in the spinal cord of nerve injured WT mice.

Conclusions/Significance

These data suggest that the increased spinal cord expression of NOS1, regulated by NOS2, might be responsible for the maintenance of chronic peripheral neuropathic pain in mice and propose these enzymes as interesting therapeutic targets for their treatment.     

The astrocyte-targeted therapy by Bushi for the neuropathic pain in mice

Background

There is accumulating evidence that the activation of spinal glial cells, especially microglia, is a key event in the pathogenesis of neuropathic pain. However, the inhibition of microglial activation is often ineffective, especially for long-lasting persistent neuropathic pain. So far, neuropathic pain remains largely intractable and a new therapeutic strategy for the pain is still required.

Methods/Principal Findings

Using Seltzer model mice, we investigated the temporal aspect of two types of neuropathic pain behaviors, i.e., thermal hyperalgesia and mechanical allodynia, as well as that of morphological changes in spinal microglia and astrocytes by immunohistochemical studies. Firstly, we analyzed the pattern of progression in the pain behaviors, and found that the pain consisted of an “early induction phase” and subsequent “late maintenance phase”. We next analyzed the temporal changes in spinal glial cells, and found that the induction and the maintenance phase of pain were associated with the activation of microglia and astrocytes, respectively. When Bushi, a Japanese herbal medicine often used for several types of persistent pain, was administered chronically, it inhibited the maintenance phase of pain without affecting the induction phase, which was in accordance with the inhibition of astrocytic activation in the spinal cord. These analgesic effects and the inhibition of astrocytic activation by Bushi were mimicked by the intrathecal injection of fluorocitrate, an inhibitor of astrocytic activation. Finally, we tested the direct effect of Bushi on astrocytic activation, and found that Bushi suppressed the IL-1β- or IL-18-evoked ERK1/2-phosphorylation in cultured astrocytes but not the ATP-evoked p38- and ERK1/2-phosphorylation in microglia in vitro.

Conclusions

Our results indicated that the activation of spinal astrocytes was responsible for the late maintenance phase of neuropathic pain in the Seltzer model mice and, therefore, the inhibition of astrocytic activation by Bushi could be a useful therapeutic strategy for treating neuropathic pain.     

Gammaherpesvirus Latency Accentuates EAE Pathogenesis: Relevance to Epstein-Barr Virus and Multiple Sclerosis

Epstein-Barr virus (EBV) has been identified as a putative environmental trigger of multiple sclerosis (MS), yet EBV''s role in MS remains elusive. We utilized murine gamma herpesvirus 68 (γHV-68), the murine homolog to EBV, to examine how infection by a virus like EBV could enhance CNS autoimmunity. Mice latently infected with γHV-68 developed more severe EAE including heightened paralysis and mortality. Similar to MS, γHV-68EAE mice developed lesions composed of CD4 and CD8 T cells, macrophages and loss of myelin in the brain and spinal cord. Further, T cells from the CNS of γHV-68 EAE mice were primarily Th1, producing heightened levels of IFN-γ and T-bet accompanied by IL-17 suppression, whereas a Th17 response was observed in uninfected EAE mice. Clearly, γHV-68 latency polarizes the adaptive immune response, directs a heightened CNS pathology following EAE induction reminiscent of human MS and portrays a novel mechanism by which EBV likely influences MS and other autoimmune diseases.