Single High-dose Injection Research Articles

Single high-dose intravenous injection of Wharton’s jelly-derived mesenchymal stem cell exerts protective effects in a rat model of metabolic syndrome

BackgroundMetabolic syndrome (MetS) is a significant epidemiological problem worldwide. It is a pre-morbid, chronic and low-grade inflammatory disorder that precedes many chronic diseases. Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) could be used to treat MetS because they express high regenerative capacity, strong immunomodulatory properties and allogeneic biocompatibility. This study aims to investigate WJ-MSCs as a therapy against MetS in a rat model.MethodsTwenty-four animals were fed with high-fat high-fructose (HFHF) diet ad libitum. After 16 weeks, the animals were randomised into treatment groups (n = 8/group) and received a single intravenous administration of vehicle, that is, 3 × 106 cells/kg or 10 × 106 cells/kg of WJ-MSCs. A healthy animal group (n = 6) fed with a normal diet received the same vehicle as the control (CTRL). All animals were periodically assessed (every 4 weeks) for physical measurements, serum biochemistry, glucose tolerance test, cardiovascular function test and whole-body composition. Post-euthanasia, organs were weighed and processed for histopathology. Serum was collected for C-reactive protein and inflammatory cytokine assay.ResultsThe results between HFHF-treated groups and healthy or HFHF-CTRL did not achieve statistical significance (α = 0.05). The effects of WJ-MSCs were masked by the manifestation of different disease subclusters and continuous supplementation of HFHF diet. Based on secondary analysis, WJ-MSCs had major implications in improving cardiopulmonary morbidities. The lungs, liver and heart show significantly better histopathology in the WJ-MSC-treated groups than in the untreated CTRL group. The cells produced a dose-dependent effect (high dose lasted until week 8) in preventing further metabolic decay in MetS animals.ConclusionsThe establishment of safety and therapeutic proof-of-concept encourages further studies by improving the current therapeutic model.

Evaluation of single and multiple hyaluronic acid injections at different concentrations with high molecular weight in the treatment of knee osteoarthritis

BackgroundKnee osteoarthritis is severe progressive and most commonly diagnosed articular disease and its incidence is increasing around the world depending on age. This pathologic condition which limits daily activity of patients can be characterized by degeneration of cartilage and inflammation. Although non-steroidal anti-inflammatory (NSAII) agents and other analgesics are routinely used treatment options, the potential effects of intraarticular injections including hyaluronic acid (HA) have also been demonstrated by various studies. However, few studies compare the efficacy of a single high molecular weight (HMW) high dose and a triple HMW low dose. This study aimed to compare the efficacy of single high molecular weight (HMW) high dose (2 mL / 60 mg) and triple HMW low dose (2 mL /30 mg) intra-articular injection of HA in knee osteoarthritis (OA) patients by evaluating function and pain parameters during 12 months.MethodsThis is a single-center, retrospective clinical study that included and involved 128 patients. Group I (n=64) patients received triple 30 mg HA injections (SEMICAL®) with one-week intervals, while Group II (n=64) patients received a single 60 mg HA injection (SEMICAL®). Lequesne Index, WOMAC and VAS scores were recorded to assess pain and function during a 12-month period.ResultsThere was no significant difference in characteristics of patient demographics. Our finding indicate that WOMAC, VAS score, and Lequesne Index values during follow-up visits exhibited a decrease, signifying improvement in the clinical condition. Notably, scores were significantly more favorable with the 30 mg of HA injection compared to the 60 mg of HA injection.ConclusionThis study suggests that the triple low-dose injection of HMW HA is more effective in improving WOMAC, VAS scores and Lequesne Index values than a single high-dose injection.

The aryl hydrocarbon receptor in β-cells mediates the effects of TCDD on glucose homeostasis in mice

ObjectiveChronic exposure to persistent organic pollutants (POPs) is associated with increased incidence of type 2 diabetes, hyperglycemia, and poor insulin secretion in humans. Dioxins and dioxin-like compounds are a broad class of POPs that exert cellular toxicity through activation of the aryl hydrocarbon receptor (AhR). We previously showed that a single high-dose injection of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, aka dioxin; 20 μg/kg) in vivo reduced fasted and glucose-stimulated plasma insulin levels for up to 6 weeks in male and female mice. TCDD-exposed male mice were also modestly hypoglycemic and had increased insulin sensitivity, whereas TCDD-exposed females were transiently glucose intolerant. Whether these effects are driven by AhR activation in β-cells requires investigation. MethodsWe exposed female and male β-cell specific Ahr knockout (βAhrKO) mice and littermate Ins1-Cre genotype controls (βAhrWT) to a single high dose of 20 μg/kg TCDD and tracked the mice for 6 weeks. ResultsUnder baseline conditions, deleting AhR from β-cells caused hypoglycemia in female mice, increased insulin secretion ex vivo in female mouse islets, and promoted modest weight gain in male mice. Importantly, high-dose TCDD exposure impaired glucose homeostasis and β-cell function in βAhrWT mice, but these phenotypes were largely abolished in TCDD-exposed βAhrKO mice. ConclusionOur study demonstrates that AhR signaling in β-cells is important for regulating baseline β-cell function in female mice and energy homeostasis in male mice. We also show that β-cell AhR signaling largely mediates the effects of TCDD on glucose homeostasis in both sexes, suggesting that the effects of TCDD on β-cell function and health are driving metabolic phenotypes in peripheral tissues.

Proinflammatory Cytokines Trigger the Onset of Retinal Abnormalities and Metabolic Dysregulation in a Hyperglycemic Mouse Model.

Recent evidence has shown that retinal inflammation is a key player in diabetic retinopathy (DR) pathogenesis. To further understand and validate the metabolic biomarkers of DR, we investigated the effect of intravitreal proinflammatory cytokines on the retinal structure, function, and metabolism in an in vivo hyperglycemic mouse model. C57Bl/6 mice were rendered hyperglycemic within one week of administration of a single high-dose intraperitoneal injection of streptozotocin, while control mice received vehicle injection. After confirming hyperglycemia, the mice received an intravitreal injection of either proinflammatory cytokines (TNF-α and IL-1β) or vehicle. Similarly, control mice received an intravitreal injection of either proinflammatory cytokines or vehicle. The retinal structure was evaluated using fundus imaging and optical coherence tomography, and retinal function was assessed using a focal electroretinogram (ERG), two days after cytokine injection. Retinas were collected for biochemical analysis to determine key metabolite levels and enzymatic activities. Hyperglycemic mice intraocularly injected with cytokines developed visible retinal vascular damage and intravitreal and intraretinal hyper-reflective spots two days after the cytokines injection. These mice also developed a significant functional deficit with reduced a-wave and b-wave amplitudes of the ERG at high light intensities compared to control mice. Furthermore, metabolic disruption was evident in these mice, with significantly higher retinal glucose, lactate, ATP, and glutamine levels and a significant reduction in glutamate levels compared with control mice. Minimal or no metabolic changes were observed in hyperglycemic mice without intraocular cytokines or in control mice with intraocular cytokines at 2 days post hyperglycemia. Proinflammatory cytokines accelerated the development of vascular damage in the eyes of hyperglycemic mice. Significant changes were observed in retinal structure, function, and metabolic homeostasis. These findings support the idea that with the onset of inflammation in DR, there is a deficit in metabolism. Therefore, early intervention to prevent inflammation-induced retinal changes in diabetic patients may improve the disease outcome.

Chronic neuroinflammation impairs waste clearance in the rat brain.

Previous reports have established an association between impaired clearance of macromolecular waste from the brain parenchyma and a variety of brain insults for which chronic neuroinflammation is a common pathological feature. Here we investigate whether chronic neuroinflammation is sufficient to impair macromolecular waste clearance from the rat brain. Using a rodent model of chronic neuroinflammation induced by a single high-dose injection of lipopolysaccharide, the clearance kinetics of two fluorophore-conjugated dextran tracers were assayed at 8-weeks post-induction. The expression and distribution of amyloid β and aquaporin-4 proteins within selected brain regions were assayed at 36-weeks post-induction, following open-field, novel object recognition, and contextual fear conditioning assays. Chronic neuroinflammation significantly impaired the clearance kinetics of both dextran tracers and resulted in significantly elevated levels of amyloid β within the hippocampus. Aquaporin-4 density on astrocytic endfeet processes was also reduced within multiple brain regions. These pathologies were associated with significantly enhanced contextual fear memory. Our results suggest that chronic neuroinflammation is sufficient to compromise the clearance of macromolecular waste from the brain parenchyma and may be the root cause of impaired waste clearance associated with a variety of brain pathologies.

Therapeutic Efficacy of Intravesical Platelet-Rich Plasma Injections for Interstitial Cystitis/Bladder Pain Syndrome-A Comparative Study of Different Injection Number, Additives and Concentrations.

Purpose: Intravesical platelet-rich plasma (PRP) injections have been demonstrated effective in relieving symptoms among patients with interstitial cystitis/bladder pain syndrome (IC/BPS). This study compared the clinical efficacy among different injection number, adding solution, and concentrations of PRP. Methods: A total of 63 patients with IC/BPS were enrolled and randomly allocated to four subgroups who received single high-dose PRP (from 100 ml whole blood) plus 10 ml of normal saline or plasma injected over 20 or 40 sites. Patients were followed up at 1, 3, and 6 months for changes in the IC symptom index (ICSI) and problem index (ICPI), visual analog scale (VAS), global response assessment (GRA), and urodynamic parameters. Furthermore, we compared the clinical outcome with our previous study in a group of 55 IC/BPS patients who underwent four monthly low-dose PRP (from 50 ml whole blood) injections. Results: The result of this study showed significant improvements in IC symptoms (ICSI 11.9 ± 4.4 vs. 10.2 ± 4.9, p = 0.009; ICPI 12.3 ± 3.4 vs. 10.6 ± 4.7, p = 0.003); VAS (5.46 ± 2.96 vs. 3.83 ± 3.1, p 0.000), and maximum flow rate (10.4 ± 4.9 vs. 17.1 ± 11.5 ml/s, p = 0.000) at 3 months after single high-dose PRP injection. However, no significant differences in therapeutic results were observed among subgroups, regardless of the added component or injecting site. The improvements of ICSI, ICPI, and GRA at 6 months were lower in comparison with the results of four low-dose PRP injections. All patients were free of dysuria, urinary retention, or urinary tract infection after PRP treatment. Conclusion: Intravesical PRP injection is effective for IC/BPS. The addition of normal saline or plasma and injection site had no influence on therapeutic efficacy. However, the symptom improvement and GRA after a single high-dose PRP injection was lower than that after four low-dose PRP injections 6 months after the first treatment. Limitation of the study is lack of sham control group.

Effectiveness of a Single High Dose of Platelet-Rich Plasma (PRP) Injection Over Corticosteroid and Hyaluronic Acid Injections on Osteoarthritis, Chronic Tendinitis and Tennis Elbow Treatment

Background: Corticosteroid, hyaluronic acid (HA) injections and Anti-inflammatory agents are considered as non-invasive treatments for knee osteoarthritis (OA), Chronic Tendinitis (CT) and Tennis elbow (TE) that are supposed to provide symptomatic relief and to help surgical delay intervention. Platelet rich plasma (PRP) is a biological component shown to be beneficial for different orthopedic dysfunctionalities treatment. The presence of GFs in PRPs such as transforming growth factor-β, insulin-like growth factor 1co-stimulate the mesenchymal stem cells and fibroblasts secretions and promotes the fibrin matrix formation which effectively drive the healing process, induces regenerative response and lead to the damage structure repair in orthopedics trauma. Methods: Three groups of a total of 30 patients presenting OA, CT and TE diagnosis, non-responding to corticosteroid, HA and non-steroid anti-inflammatory treatments were randomized to undergo one intra-articular injections of single high dose of PRP. The efficacy of Intra-articular PRP Injections was evaluated before the injection and one month after. The efficiency assessment score was based on [1] Knee injury and Osteoarthritis Outcome Score, [2] Physical Function Short Form (KOOS-PS) Arabic (KSA) version LK 1.0, [3] HOOS-Physical Function Short form (HOOS-PS), and [4] Macdermid patient-rated Tennis Elbow. Results: A significant reduction of pain and a marked improvement in movements was observed in the 3 patient’s groups, PRP-injected patients showed significantly higher values compared with baseline: (p < 0.005 vs baseline), improve functional status and reduce clearly the articular dysfunctions over the time. In our study, single High dose injection of PRP provided an overall superior clinical improvement compared with HA and corticosteroid treatments over the time and the different follow-up checkpoints of the study.

Investigating the role of Ca2+/calmodulin-dependent protein kinase II in the survival of retinal ganglion cells.

Investigating the role of Ca2+/calmodulin-dependent protein kinase II in the survival of retinal ganglion cells.

Abnormal electrophysiological phenotypes and sleep deficits in a mouse model of Angelman Syndrome

BackgroundAngelman Syndrome (AS) is a rare genetic disorder characterized by impaired communication, motor and balance deficits, intellectual disabilities, recurring seizures and abnormal sleep patterns. The genetic cause of AS is neuronal-specific loss of expression of UBE3A (ubiquitin-protein ligase E6-AP), an imprinted gene. Seizure and sleep disorders are highly prevalent (> 80%) in the AS population. The present experiments were designed to identify translational, neurophysiological outcome measures in a model of AS.MethodsWe used the exon-2 deletion mouse (Ube3a-del) on a C57BL/6J background to assess seizure, sleep and electrophysiological phenotypes. Seizure susceptibility has been reported in Ube3a-del mice with a variety of seizure induction methods. Here, we provoked seizures by a single high-dose injection of 80 mg/kg pentylenetetrazole. Novel experiments included the utilization of wireless telemetry devices to acquire global electroencephalogram (EEG) and neurophysiological data on electrographic seizures, power spectra, light–dark cycles, sleep stages and sleep spindles in Ube3a-del and WT mice.ResultsUbe3a-del mice exhibited reduced seizure threshold compared to WT. EEG illustrated that Ube3a-del mice had increased epileptiform spiking activity and delta power, which corroborates findings from other laboratories and recapitulates clinical reports in AS. This is the first report to use a cortical surface-based recording by a wireless telemetry device over tethered/fixed head-mount depth recordings. Less time in both paradoxical and slow-wave sleep, longer latencies to paradoxical sleep stages and total less sleep time in Ube3a-del mice were observed compared to WT. For the first time, we detected fewer sleep spindles in the AS mouse model.LimitationsThis study was limited to the exon 2 deletion mouse model, and future work will investigate the rat model of AS, containing a complete Ube3a deletion and pair EEG with behavior.ConclusionsOur data enhance rigor and translatability as our study provides important corroboration of previous reports on epileptiform and elevated delta power. For the first time we report neurophysiological phenotypes collected via translational methodology. Furthermore, this is the first report of reduced sleep spindles, a critical marker of memory consolidation during sleep, in an AS model. Our results are useful outcomes for therapeutic testing.

Long-term metabolic consequences of acute dioxin exposure differ between male and female mice

Epidemiological studies have consistently shown an association between exposure to environmental pollutants and diabetes risk in humans. We have previously shown that direct exposure of mouse and human islets (endocrine pancreas) to the highly persistent pollutant TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) causes reduced insulin secretion ex vivo. Furthermore, a single high-dose of TCDD (200 µg/kg) suppressed both fasting and glucose-induced plasma insulin levels and promoted beta-cell apoptosis after 7 days in male mice. The current study investigated the longer-term effects of a single high-dose TCDD injection (20 µg/kg) on glucose metabolism and beta cell function in male and female C57Bl/6 mice. TCDD-exposed males displayed modest fasting hypoglycemia for ~4 weeks post-injection, reduced fasting insulin levels for up to 6 weeks, increased insulin sensitivity, decreased beta cell area, and increased delta cell area. TCDD-exposed females also had long-term suppressed basal plasma insulin levels, and abnormal insulin secretion for up to 6 weeks. Unlike males, TCDD did not impact insulin sensitivity or islet composition in females, but did cause transient glucose intolerance 4 weeks post-exposure. Our results show that a single exposure to dioxin can suppress basal insulin levels long-term in both sexes, but effects on glucose homeostasis are sex-dependent.

Functional cytochrome P450 1A enzymes are induced in mouse and human islets following pollutant exposure

Aims/hypothesisExposure to environmental pollution has been consistently linked to diabetes incidence in humans, but the potential causative mechanisms remain unclear. Given the critical role of regulated insulin secretion in maintaining glucose homeostasis, environmental chemicals that reach the endocrine pancreas and cause beta cell injury are of particular concern. We propose that cytochrome P450 (CYP) enzymes, which are involved in metabolising xenobiotics, could serve as a useful biomarker for direct exposure of islets to pollutants. Moreover, functional CYP enzymes in islets could also impact beta cell physiology. The aim of this study was to determine whether CYP1A enzymes are activated in islets following direct or systemic exposure to environmental pollutants.MethodsImmortalised liver (HepG2) and rodent pancreatic endocrine cell lines (MIN6, βTC-6, INS1, α-TC1, α-TC3), as well as human islets, were treated in vitro with known CYP1A inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene (3-MC). In addition, mice were injected with either a single high dose of TCDD or multiple low doses of TCDD in vivo, and islets were isolated 1, 7 or 14 days later.ResultsCYP1A enzymes were not activated in any of the immortalised beta or alpha cell lines tested. However, both 3-MC and TCDD potently induced CYP1A1 gene expression and modestly increased CYP1A1 enzyme activity in human islets after 48 h. The induction of CYP1A1 in human islets by TCDD was prevented by cotreatment with a cytokine mixture. After a systemic single high-dose TCDD injection, CYP1A1 enzyme activity was induced in mouse islets ~2-fold, ~40-fold and ~80-fold compared with controls after 1, 7 and 14 days, respectively, in vivo. Multiple low-dose TCDD exposure in vivo also caused significant upregulation of Cyp1a1 in mouse islets. Direct TCDD exposure to human and mouse islets in vitro resulted in suppressed glucose-induced insulin secretion. A single high-dose TCDD injection resulted in lower plasma insulin levels, as well as a pronounced increase in beta cell death.Conclusions/interpretationTransient exposure to TCDD results in long-term upregulation of CYP1A1 enzyme activity in islets. This provides evidence for direct exposure of islets to lipophilic pollutants in vivo and may have implications for islet physiology.

Resveratrol solid lipid nanoparticles to trigger credible inhibition of doxorubicin cardiotoxicity.

Background: Doxorubicin (DOX), a broad-spectrum chemotherapy drug, is clinically employed to treat cancers especially for breast cancer and lung cancer. But its clinical applications are limited by the dose-dependent cardiac toxicity. Resveratrol (Res), a polyphenolic antitoxin, has been proved to be capable of improving the cardiomyocyte calcium cycling by up-regulating SIRT-1-mediated deacetylation to inhibit DOX-induced cardiotoxicity.Purpose: The objective of this study was to develop a solid lipid nanoparticle (SLN) loaded with Res to trigger inhibition of DOX-induced cardiotoxicity.Methods: Res-SLN was prepared by emulsification-diffusion method followed by sonication and optimized using central composite design/response surface method. The Res-SLN was further evaluated by dynamic light scattering, transmission electron microscopy for morphology and high performance liquid chromatography for drug loading and release profile. And the Res distribution in vivo was determined on rats while the effect of inhibit DOX-induced cardiotoxicity was investigated on mice.Results: Res-SLN with homogeneous particle size of 271.13 nm was successfully formulated and optimized. The prepared Res-SLN showed stable under storage and sustained release profile, improving the poor solubility of Res. Heart rate, ejection fractions and fractional shortening of Res-SLN treating mice were found higher than those on mice with cardiac toxicity induced by single high-dose intraperitoneal injection of DOX. And the degree of myocardial ultrastructural lesions on mice was also observed.Conclusion: Res-SLN has a certain therapeutic effect for protecting the myocardium and reducing DOX-induced cardiotoxicity in mice.

The effects of a single high-dose injection of vitamin-D on healing of condylar fractures in osteoporotic rats

The effects of a single high-dose injection of vitamin-D on healing of condylar fractures in osteoporotic rats

Muscle loss following a single high-dose intramuscular injection of corticosteroids to treat disease flare in patients with rheumatoid arthritis.

Adverse changes in body composition, specifically decreased muscle mass (MM) and increased fat mass, characterize rheumatoid arthritis (RA). These changes, termed rheumatoid cachexia (RC), are important contributors to the disability and elevated co-morbidity risk of RA. Recently, we observed substantial muscle loss (~2 kg) in a patient with RA following a single intramuscular (IM) corticosteroid (CS) injection to treat a disease flare. The aim of the current study is to determine whether this apparent iatrogenic effect of IM CS is typical, i.e., does this routine, recommended treatment contribute to RC? Body composition was assessed by dual-energy X-ray absorptiometry (DXA) in eight patients with established RA who received a 120 mg IM methylprednisolone injection to treat a disease flare. DXA scans estimated appendicular lean mass (ALM; a surrogate measure of MM), total lean mass (LM), and total and regional adiposity at baseline (injection day) and 4 weeks and 6-9 months post-injection. Statistical analysis was performed using one-way ANOVA. There was significant loss of ALM (-0.93 kg, p=0.001, 95% CI [-0.49, -1.36]) and a trend toward reduced LM (-1.10 kg, p=0.165, 95% CI [0.58, -2.79]) at 4 weeks relative to baseline. At 6-9 months despite control of inflammation and disease activity, these losses remained. Substantial muscle loss occurred in patients with RA following IM CS injection to treat a disease flare. Thus, this recommended treatment appears to exacerbate RC, thereby potentially increasing disability and co-morbidity risk. If this effect is confirmed by larger studies, the role of one-off high-dose CS in the treatment of RA should be reviewed.

Tendinopathy Induced by Serial Low-Dose Collagenase Injections

Category: Basic Sciences/Biologics Introduction/Purpose: Numerous studies, as evident in literature, have attempted to develop an induced Achilles tendinopathy animal model. The most common models include collagenase injections and mechanical overload of the tendon, but both are controversial. The mechanical overload model creates a chronically induced tendinopathy and is time intensive. In addition, once the stimulus is removed the tissue changes often heal. The collagenase injection model is quicker, rarely evolve to healing but frequently results in an early acute tendon reaction. The primary objective of this study is to compare biomechanical and histological findings between two collagenase induced Achilles tendinopathy protocols. The hypothesis is that consecutive low-dose collagenase injections will result in progressive and long-lasting tendinopathy findings as compared to the traditional single high-dose injection. Methods: In this IRB approved study, the population was composed of forty-eight (n=48) New Zealand breed rabbits. Forty-two (n=42) rabbits were randomly divided into two groups (n=21). The first group, which served as the control, had both Achilles tendons injected by a single dose (0.3 mg) of A1 collagenase (Sigma-Aldrich©), as described in literature. The second experimental group had three low-dose injections (0.10 mg) with 2 weeks between each injection. Another six animals (n=6) were also randomized into two groups (n=3) receiving one-dose versus 3 injections of saline solution with 2 weeks between each injection. The animals were euthanized after 10, 12 and 16 weeks. Histological and biomechanic analysis of the Achilles tendons were carried out using a dynamic mechanical testing machine (Electropuls®, model E10000, Instron®). Mechanical strength and histological scores of tendinopathy (Bonar scoring system) were compared among the groups at each time-point. Results: After 16 weeks, all biomechanical and histological parameters analysed showed consistent differences between the groups (p &lt; 0.05), with more pronounced and long-lasting tendinopathy findings in the Achilles tendon of the experimental group (serial collagenase injections), when compared to the control groups (single collagenase dose and single/multiple saline injections). The mean Bonar sum-score of tendons after 16 weeks in the experimental group was greater than the mean histologic score of control tendons (8.53 ± 1.52 versus 1.1 ± 0.83 in saline group and 5.2 ± 1.12 in single dose group). No statistically significant differences were found between the different collagenase injection groups at weeks 10 and 12. Conclusion: The protocol of three consecutive low-dose collagenase injections has shown biomechanical and histological findings compatible with progressive and long-lasting tendinopathy as compared to single high-dose injection. This protocol represents a feasible and effective animal model of induced Achilles tendinopathy.

Elusive sources of variability of dystrophin rescue by exon skipping.

BackgroundSystemic delivery of anti-sense oligonucleotides to Duchenne muscular dystrophy (DMD) patients to induce de novo dystrophin protein expression in muscle (exon skipping) is a promising therapy. Treatment with Phosphorodiamidate morpholino oligomers (PMO) lead to shorter de novo dystrophin protein in both animal models and DMD boys who otherwise lack dystrophin; however, restoration of dystrophin has been observed to be highly variable. Understanding the factors causing highly variable induction of dystrophin expression in pre-clinical models would likely lead to more effective means of exon skipping in both pre-clinical studies and human clinical trials.MethodsIn the present study, we investigated possible factors that might lead to the variable success of exon skipping using morpholino drugs in the mdx mouse model. We tested whether specific muscle groups or fiber types showed better success than others and also correlated residual PMO concentration in muscle with the amount of de novo dystrophin protein 1 month after a single high-dose morpholino injection (800 mg/kg). We compared the results from six muscle groups using three different methods of dystrophin quantification: immunostaining, immunoblotting, and mass spectrometry assays.ResultsThe triceps muscle showed the greatest degree of rescue (average 38±28 % by immunostaining). All three dystrophin detection methods were generally concordant for all muscles. We show that dystrophin rescue occurs in a sporadic patchy pattern with high geographic variability across muscle sections. We did not find a correlation between residual morpholino drug in muscle tissue and the degree of dystrophin expression.ConclusionsWhile we found some evidence of muscle group enhancement and successful rescue, our data also suggest that other yet-undefined factors may underlie the observed variability in the success of exon skipping. Our study highlights the challenges associated with quantifying dystrophin in clinical trials where a single small muscle biopsy is taken from a DMD patient.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0070-6) contains supplementary material, which is available to authorized users.

Repeated low doses of morpholino antisense oligomer: an intermediate mouse model of spinal muscular atrophy to explore the window of therapeutic response.

The human SMN2 transgenic mice are well-established models of spinal muscular atrophy (SMA). While the severe type I mouse model has a rapidly progressive condition mimicking type I SMA in humans, the mild type III mice do not faithfully recapitulate chronic SMA variants affecting children. A SMA mouse model that clinically mimics the features of type II and III SMA in human is therefore needed. In this study, we generated intermediately affected SMA mice by delivering low-dose morpholino oligomer (PMO25) into the existing severe SMA mice. We show that a single low-dose administration of PMO25 moderately extended the survival of severe type I SMA mice. The neuromuscular pathology is also modestly but significantly improved in these mice. A second administration of PMO25 at postnatal day 5 (PND5) demonstrated an additive effect on survival. Additional systemic administration of low-dose PMO25 at 2-week intervals suppressed the occurrence of distal necrosis beyond postnatal day 100, and induced more complete phenotypic rescue than a single bolus high-dose injection at PND0. Our study demonstrates that survival of motor neuron (SMN) is required early at a critical threshold to prevent symptoms and suggests that subsequent systemic administration of low-dose PMO25 in SMA mice can provide therapeutic benefit and phenotypic rescue, presumably via peripheral SMN restoration. Our work also provides additional insight into the time window of response to administration of antisense oligonucleotides to SMA mice with an intermediate phenotype. This information is crucial at a time when a number of therapeutic interventions are in clinical trials in SMA patients.

Fibroblast growth factor 21 prevents glycemic deterioration in insulin deficient mouse models of diabetes

In type 1 diabetes, there is a rapid loss of glycemic control immediately after onset of the disease. We aimed to determine if the deterioration of glycemic control that occurs early after the onset of insulin-deficient diabetes could be blunted by treatment with recombinant fibroblast growth factor 21 (FGF21). Normal C57BL/6J mice made diabetic by a single high dose injection of streptozotocin (STZ) were randomized to receive twice daily subcutaneous injection of vehicle or recombinant human FGF21 at doses of 0.3 and 1.0mg/kg for 10 days. Body weight was recorded daily and 5h fasted glucose, insulin, glucagon, free fatty acids and ketones were determined at 6 and 10 days post-randomization.The increase in fasting plasma glucose induced by STZ in untreated mice was prevented with FGF21 at 0.3mg/kg BID. In contrast, at 1.0mg/kg BID, FGF21 did not prevent the rise in plasma glucose after STZ. At the end of the study, plasma glucagon was significantly higher in the diabetic group treated with FGF21 1.0mg/kg BID than in the untreated group. This was not seen for the group treated with FGF21 0.3mg/kg BID. There were significant dose dependent reductions in plasma free fatty acids with FGF21 treatment but no significant change in plasma ketones (β-hydroxybutyrate). FGF21 treatment did not have significant effects on body weight in lean insulin deficient mice.In conclusion, FGF21 prevents increases in glycaemia and has lipid lowering properties in mouse models of insulin deficient diabetes, although by increasing the dose increased glucagon levels are seen and hyperglycemia persists.

BDNF modifies hippocampal KCC2 and NKCC1 expression in a temporal lobe epilepsy model.

Excitatory GABA actions, induced by altered expression of chloride transporters (KCC2/NKCC1), can contribute to seizure generation in temporal lobe epilepsy. In the present study, we evaluated whether BDNF administration can affect KCC2/NKCC1 expression, ictogenesis and behavioral alterations in this paradigm. Status epilepticus was induced in male rats with pilocarpine, followed by a treatment of either a single high dose or multiple injections of BDNF during the latent phase of temporal lobe epilepsy. Chloride transporters expression, spontaneous recurrent seizures, and hyperexcitability post-seizural behaviors were evaluated after treatment. NKCC1 protein expression was markedly upregulated, whereas that of KCC2 was significantly downregulated in epileptic hippocampi compared to intact controls. Application of BDNF (both single high dose and multiple injections) increased KCC2 expression in epileptic hippocampi, while NKCC1 expression was downregulated exclusively by the single high dose injection of BDNF. Development of spontaneous recurrent seizures was delayed but not prevented by the treatment, and hyperexcitability behaviors were ameliorated for a short period of time. To prevent GABA-A mediated depolarization and design appropriate treatment strategies for temporal lobe epilepsy, chloride transporters can be considered as a target. Future studies are warranted to investigate any possible therapeutic effects of BDNF via altering chloride transporters expression.

CT-guided single high-dose percutaneous acetic acid injection for small hepatocellular carcinoma: A long-term follow-up study

To evaluate the long-term outcome of CT-guided single high-dose percutaneous acetic acid injection (PAI) for the treatment of small hepatocellular carcinoma (SHCC). Sixty-eight consecutive patients (37 men and 31 women; age 52 ± 16 years) with SHCC have undergone CT-guided single high-dose PAI therapy since December 2000. Complications of high-dose PAI were recorded. Tumour survival and recurrence rates were assessed. All PAI therapies were successful and no severe complications developed. The average follow-up period was 46 ± 18 months. The overall survival rates at 1, 2, 3, 4 and 5 years were 93%, 82%, 68%, 59% and 51%, respectively. The cumulative local recurrence rates for the main tumour at 1, 2, 3, 4 and 5 years were 18%, 22%, 25%, 28% and 28%, respectively. The cumulative new tumour recurrence rates at 1, 2, 3, 4 and 5 years were 24%, 40%, 43%, 60% and 62%, respectively. CT-guided single high-dose PAI is safe and effective for the treatment of SHCC after long-term follow-up.