Post-injection Day Research Articles

Efficacy and Safety of Concurrent Collagenase Clostridium Histolyticum Injections for Multiple Dupuytren Contractures

To assess the safety and efficacy of 2 concurrent injections of collagenase clostridiumhistolyticum (CCH) in the same hand to treat multiple Dupuytren flexion contractures. In a multicenter, open-label phase IIIb study, 60 patients received two 0.58-mg CCHdoses injected into cords affecting 2 joints in the same hand during 1 visit, followed by finger extension approximately 24 hours later. Efficacy at postinjection day 30 (change in flexion contracture and active range of motion, patient satisfaction, physician-rated improvement, and rates of clinical success [flexion contracture 5° or less]) and adverse events were summarized. The concurrent injections were most commonly administered in cords affecting metacarpophalangeal (MCP) and proximal interphalangeal (PIP) joints on the same finger (47%) or 2 MCP joints on different fingers of the same hand (37%). Mean total (sum of the 2 treated joints) flexion contracture decreased 76%, from 87° to 24° (MCP joints: 86%; PIP joints: 66%). Mean total range of motion increased from 100° to 161°. Clinical success was 76% for MCP joints and 33% for PIP joints. Most patients were very satisfied (60%) or quite satisfied (28%) with treatment. Most investigators rated treated joints as very much improved (55%) or much improved (37%). The most common treatment-related adverse events (> 75% of patients) were contusion, pain in extremity, and edema peripheral (local edema). Most adverse events were mild to moderate in severity. Serious complications included 1 pulley rupture related to study medication and 1 flexor tendon rupture (following conclusion of the study). There were no systemic complications. Results suggest that 2 affected joints can be effectively and safely treated with concurrent CCH injections. There was an increased incidence of some adverse events with concurrent treatment (pruritus, lymphadenopathy, blood blister, and skin laceration) compared with treatment of a single joint. High degrees of patient satisfaction and physician-rated improvement were reported. Therapeutic IV.

Intravitreal Injection of Ranibizumab for Treatment of Age-Related Macular Degeneration: Effects on Serum VEGF Concentration

Aims: To evaluate potential adverse ranibizumab-related systemic events through analysis of variations in serum levels of vascular endothelial growth factor (VEGF) in neovascular age-related macular degeneration (AMD) patients before and after a single intravitreal injection of ranibizumab.Methods: Thirty-nine patients with neovascular AMD and 39 healthy control subjects were enrolled in the study. Patients received a single intravitreal injection of ranibizumab (0.5 mg) in one eye. Venous blood was collected and placed in coagulation-promoting tubes 1 day before and on post-injection days 1, 3, 7 and 30. Serum concentrations of VEGF were measured by ELISA at each time point.Results: VEGF concentrations were 323.64 pg/ml in AMD patients and 392.94 pg/ml in control subjects before injection (p > 0.05). VEGF significantly decreased to 304.65 pg/ml 1 day later (p < 0.05) in AMD patients, then increased to 310.77 (p > 0.05), 317.89 (p > 0.05) and 311.79 pg/ml (p > 0.05) on post-injection days 3, 7 and 30, respectively.Conclusion: No significant changes in serum levels of VEGF were found from 3 to 30 days following a single intravitreal ranibizumab injection. Although certain influences existed 24-h post-injection, effect(s) of a single intravitreal ranibizumab injection on the homeostasis of the cardiovascular system during such a brief period is unknown.

Effect of thimerosal on the neurodevelopment of premature rats

This study was undertaken to determine the effect of thimerosal on the neurodevelopment of premature rats. Thimerosal was injected into premature SD rats at a dose of 32.8, 65.6, 98.4 or 131.2 μg/kg on postnatal day 1. Expression of dopamine D4 receptor (DRD4) and serotonin 2A receptor (5-HT2AR), apoptosis in the prefrontal cortex on post-injection day 49, and learning and memory function were studied and compared with those in a control group injected with saline. Expression of DRD4 and 5-HT2AR and learning function decreased, and apoptosis increased significantly in the 131.2 μg/kg group (P<0.001). Memory function was significantly impaired by 65.6 (P<0.05), 98.4 and 131.2 μg/kg (P<0.001). The negative adverse consequences on neurodevelopment observed in the present study are consistent with previous studies; this study raised serious concerns about adverse neurodevelopmental disorder such as autism in humans following the ongoing worldwide routine administration of thimerosalcontaining vaccines to infants.

Pharmacokinetics of subcutaneous depot medroxyprogesterone acetate injected in the upper arm

BackgroundThe abdomen and thigh are recommended injection sites in the label for Depo-SubQ Provera 104™. We evaluated the pharmacokinetic profile of medroxyprogesterone acetate (MPA) following injection of Depo-SubQ Provera 104 in the upper arm, a preferred injection site in developing countries. Study designTwenty-six women in Norfolk, VA, received a single injection of Depo-SubQ Provera 104 in the upper arm in this prospective noncomparative study. We measured MPA serum concentrations prior to injection (day 1) and 11 times postinjection (days 2, 4, 8, 14, 30, 44, 60, 74, 91, 104 and 120). ResultsSerum MPA levels peaked at 0.953 ng/mL 2–14 days (interquartile range; median=8) after dosing. Mean AUC0–91 was 45.1 ng·day/mL. Mean MPA levels at days 91, 104 and 120 were 0.427, 0.367 and 0.327 ng/mL, respectively. A total of 15 individual measurements of MPA were below 0.2 ng/mL. All women but one had MPA levels above 0.1 ng/mL on day 91. ConclusionsInjection of Depo-SubQ Provera 104™ in the upper arm provided sufficient MPA levels for contraceptive protection for 3 months (13 weeks). The uptake and metabolism of MPA when injected in the upper arm may be different from the abdomen and thigh.

Characterization of glial fibrillary acidic protein (GFAP)-expressing hepatic stellate cells and myofibroblasts in thioacetamide (TAA)-induced rat liver injury

Hepatic stellate cells (HSCs), which can express glial fibrillary acidic protein (GFAP) in normal rat livers, play important roles in hepatic fibrogenesis through the conversion into myofibroblasts (MFs). Cellular properties and possible derivation of GFAP-expressing MFs were investigated in thioacetamide (TAA)-induced rat liver injury and subsequent fibrosis. Seven-week-old male F344 rats were injected with TAA (300mg/kg BW, once, intraperitoneally), and were examined on post single injection (PSI) days 1–10 by the single and double immunolabeling with MF and stem cell marker antibodies. After hepatocyte injury in the perivenular areas on PSI days 1 and 2, the fibrotic lesion consisting of MF developed at a peak on PSI day 3, and then recovered gradually by PSI day 10. MFs expressed GFAP, and also showed co-expressions such cytoskeletons (MF markers) as vimentin, desmin and α-SMA in varying degrees. Besides MFs co-expressing vimentin/desmin, desmin/α-SMA or α-SMA/vimentin, some GFAP positive MFs co-expressed with nestin or A3 (both, stem cell markers), and there were also MFs co-expressing nestin/A3. However, there were no GFAP positive MFs co-expressing RECA-1 (endothelial marker) or Thy-1 (immature mesenchymal cell marker). GFAP positive MFs showed the proliferating activity, but they did not undergo apoptosis. However, α-SMA positive MFs underwent apoptosis. These findings indicate that HSCs can proliferate and then convert into MFs with co-expressing various cytoskeletons for MF markers, and that the converted MFs may be derived partly from the stem cell lineage. Additionally, well-differentiated MFs expressing α-SMA may disappear by apoptosis for healing. These findings shed some light on the pathogenesis of chemically induced hepatic fibrosis.

SaRNA Guided iNOS Up-Regulation Improves Erectile Function of Diabetic Rats

Promoter targeted saRNAs mediate sequence specific up-regulation of gene expression. We explored the therapeutic effect of RNA activation mediated iNOS gene activation on improving erectile function in a rat model of diabetes mellitus. An optimal saRNA sequence specific for iNOS promoter was cloned into an adenoviral vector, resulting in AdU6/shiNOS and AdU6/shControl. The corresponding viruses were used to transduce cultured rat cavernous smooth muscle cells. Streptozotocin induced diabetes models were established in rats and used to test the effects of intracavernous delivery of iNOS saRNA viruses on erectile function. iNOS expression in the cavernous smooth muscle cells or penile tissue of treated rats was assessed by reverse transcriptase-polymerase chain reaction and Western blot. Cyclic guanosine monophosphate was analyzed by enzyme-linked immunosorbent assay. Intracavernous pressure in response to cavernous nerve stimulation was measured using a data acquisition system on post-injection days 1, 3, 5, 7, 10 and 14. Adenovirus mediated expression of iNOS saRNA caused sustained up-regulation of iNOS in cavernous smooth muscle cells. Intracavernous injection of AdU6/shiNOS activated iNOS expression in vivo and significantly increased peak intracavernous pressure in streptozotocin induced diabetic rats via nitric oxide/intracellular cyclic guanosine monophosphate activation. Results show that saRNA mediated iNOS over expression in the penis can restore erectile function in streptozocin diabetic rats via the nitric oxide-cyclic guanosine monophosphate pathway.

Echinococcus multilocularis infection leads to increased expression of proliferation and anti-apoptosis factors in liver cells in BALB/c mice

To investigate the effects of Echinococcus multilocularis on host liver cell proliferation in vivo using a BALB/c mouse alveolar hydatid infection model. Sixty-five 8-10-week-old female BALB/c mice were randomly divided into an experimental group (n = 40) and a control group (n = 25) and administered an abdominal injection into the left liver lobe of E. multilocularis protoscolices in saline solution or saline solution alone, respectively. At post-injection day 2, 8, 30, 60, and 90, liver samples were collected for analysis of lesions and lesion-adjacent tissue by hematoxylin-eosin staining and differential expression of proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin A, and cyclin B1 by immunohistochemical staining. The significance of intergroup differences was assessed by Student's t-test. The control group showed normal liver histology at all time points. The experimental group developed E. multilocularis lesions that showed increased severity of pathological features, such as inflammatory cell invasion, steatosis and fibrous connective tissue hyperplasia, over time. At post-injection days 2 and 8, enlarged, binuclear and apocyte hepatocytes were observed close to the lesions. At post-injection days 30, 60, and 90, the number of hepatocytes expressing PCNA progressively increased in the experimental group, and the numbers were significantly higher than in the control group (7.01 +/- 1.89 vs. 1.03 +/- 0.52, 8.41 +/- 2.80 vs. 0.93 +/- 0.31, and 13.4 +/- 4.43 vs. 1.07 +/- 0.94; all P < 0.05). The same progressively increasing trend was seen in the number of hepatocytes expressing CyclinD1, but was only significantly different from controls at post-injection days 30 and 60 (6.73 +/- 2.52 vs. 0.48 +/- 0.43 and 8.22 +/- 3.09 vs. 0.55 +/- 0.34; both P < 0.05). In contrast, the number of hepatocytes expressing cyclin A was significantly increased at post-injection day 30 and then showed a decreasing trend at days 60 and 90, although the numbers of expressing cells remained significantly higher than control levels at all time points (7.75 +/- 3.05 vs. 0.69 +/- 0.36, 3.42 +/- 1.80 vs. 1.14 +/- 0.42, and 3.03 +/- 1.50 vs. 0.69 +/- 0.31; all P < 0.05). The number of hepatocytes expressing CyclinB1 in the experimental group was less robust than the other cyclins (with a general temporal trend of increase followed by decrease), but the differential expression was not significantly different from the control levels at any time point. E. multilocularis infection may promote the expression of host factors related to proliferation and anti-apoptosis in liver. This pathogen-mediated modulation of host cell-survival mechanisms may provide a rationale explanation for the clinical observations of hepatomegaly and the unexpected survival of alveolar echinococcosis patients following major hepatic resection.

Knockdown of orexin type 2 receptor in the lateral pontomesencephalic tegmentum of rats increases REM sleep

Dysfunction of the orexin/hypocretin neurotransmitter system causes the sleep disorder narcolepsy, characterized by intrusion of rapid eye movement (REM) sleep-like events into normal wakefulness. The sites where orexins act to suppress REM sleep are incompletely understood. Previous studies suggested that the lateral pontomesencephalic tegmentum (lPMT) contains an important REM sleep inhibitory area, and proposed that orexins inhibit REM sleep via orexin type 2 receptors (OxR2) in this region. However, this hypothesis has heretofore not been tested. We thus performed bilateral injection of small interfering RNAs (siRNAs) targeting Ox2R into the lPMT on two consecutive days. This led to a approximately 30% increase of time spent in REM sleep in both the dark and light periods for the first 2 days after injection, with a return to baseline over the next two post-injection days. This increase was mainly due to longer (> 120 s) REM episodes. Cataplexy-like episodes were not observed. The percentage of time spent in wakefulness and non-(N)REM sleep, as well as the power spectral profile of NREM and REM sleep, were unaffected. Control animals injected with scrambled siRNA had no sleep changes post-injection. Quantification of the knockdown revealed that unilateral microinjection of siRNAs targeting OxR2 into the lPMT induced a approximately 40% reduction of OxR2 mRNA 2 days following the injections when compared with the contralateral side receiving control (scrambled) siRNA. Orexin type 1 receptor mRNA level was unaffected. Our results indicate that removal of OxR2 neurotransmission in the lPMT enhances REM sleep by increasing the duration of REM episodes.

Comparative Study of Biological Activity of Four Botulinum Toxin Type A Preparations in Mice

Units of available botulinum toxin preparations are not interchangeable, and the dose-conversion ratios between such preparations remain controversial. To compare the efficacy and safety of four botulinum toxin type A preparations. Murine gastrocnemius compound muscle action potentials (CMAPs) were recorded before and after injecting the four botulinum toxin preparations (onabotulinumtoxinA, abobotulinumtoxinA, new botulinum toxin, and incobotulinumtoxinA). In all preparations, CMAP amplitudes decreased until 4days after receiving the injection and then gradually recovered. On postinjection day 84, the amplitudes returned to baseline in all groups except the high-dose groups. CMAP amplitude in the contralateral limb also decreased up to postinjection days 4 to 7 and then gradually returned to baseline by postinjection day 28. The dose-conversion ratio between onabotulinumtoxinA and abobotulinumtoxinA was determined to be 1:2.6; previous reports of 1:3 were considered too high. A dose-conversion ratio between onabotulinumtoxinA and new botulinum toxin of 1:1 was deemed appropriate. OnabotulinumtoxinA and incobotulinumtoxinA demonstrated a dose-conversion ratio of 1:1.07. The efficacy of incobotulinumtoxinA was slightly lower than that of onabotulinumtoxinA. These dose-conversion ratios are applicable solely from an efficacy standpoint and not for safety. This study was conducted in mice, so it may not translate perfectly to human applications.

Single and multiple injections of subconjunctival ranibizumab for early, recurrent pterygium

PurposeTo assess the effect of single versus multiple subconjunctival ranibizumab injections in patients with an early pterygium recurrence.SettingSingle-center, academic practice.Study populationNine patients with early pterygium recurrence.Observational procedureSubconjunctival ranibizumab (0.5 mg/0.05 mL) was administered adjacent to pterygium recurrence. Group 1 (n = 5) received one injection; group 2 (n = 4) received three injections (time points 0, 2, and 4 weeks) with the ability to retreat as needed.Main outcome measuresEffect of ranibizumab on conjunctival hyperemia and corneal neovascular area over a 6-month follow-up period.ResultsIn the single injection group, a decrease in conjunctival hyperemia was noted in all patients on postinjection day 1. At follow up, hyperemia grade fluctuated, although all patients had less hyperemia than at baseline. In the recurrent injection group, the median number of injections was 8.5 (range 7 to 9) over the 6 months. In spite of the repeated injections, the pattern of conjunctival hyperemia was similar to that of the single injection group. In group 1, corneal neovascularization remained relatively unchanged over the 6-month period in four patients and decreased in one patient by 24%. In group 2, corneal neovascularization increased in one patient by 39%, remained stable in one patient, and decreased in two patients by 34% and 44%.ConclusionThis is the first study to evaluate the role of ranibizumab in the treatment of an early pterygium recurrence and the first to compare multiple versus single injections. Recurrent injections did not appear to be superior to a single injection with regards to conjunctival hyperemia.

Intervention of Electroacupuncture on Spinal p38 MAPK/ATF-2/VR-1 Pathway in Treating Inflammatory Pain Induced by CFA in Rats

BackgroundPrevious studies have demonstrated that p38 MAPK signal transduction pathway plays an important role in the development and maintenance of inflammatory pain. Electroacupuncture (EA) can suppress the inflammatory pain. However, the relationship between EA effect and p38 MAPK signal transduction pathway in inflammatory pain remains poorly understood. It is our hypothesis that p38 MAPK/ATF-2/VR-1 and/or p38 MAPK/ATF-2/COX-2 signal transduction pathway should be activated by inflammatory pain in CFA-injected model. Meanwhile, EA may inhibit the activation of p38 MAPK signal transduction pathway. The present study aims to investigate that anti-inflammatory and analgesic effect of EA and its intervention on the p38 MAPK signal transduction pathway in a rat model of inflammatory pain.ResultsEA had a pronounced anti-inflammatory and analgesic effect on CFA-induced chronic inflammatory pain in rats. EA could quickly raise CFA-rat’s paw withdrawal thresholds (PWTs) and maintain good and long analgesic effect, while it subdued the ankle swelling of CFA rats only at postinjection day 14. EA could down-regulate the protein expressions of p-p38 MAPK and p-ATF-2, reduced the numbers of p-p38 MAPK-IR cells and p-ATF-2-IR cells in spinal dorsal horn in CFA rats, inhibited the expressions of both protein and mRNA of VR-1, but had no effect on the COX-2 mRNA expression.ConclusionsThe present study indicates that inhibiting the activation of spinal p38 MAPK/ATF-2/VR-1 pathway may be one of the main mechanisms via central signal transduction pathway in the process of anti-inflammatory pain by EA in CFA rats.

Expressions of Iba1 and galectin-3 (Gal-3) in thioacetamide (TAA)-induced acute rat liver lesions

Ionized calcium binding adaptor molecule 1 (Iba1) is associated with membrane ruffling and motility of cells. Galectin-3 (Gal-3) is a β-galactoside binding animal lectin, and regulates fibrogenesis probably through transforming growth factor-β1. To evaluate macrophage properties, expressions of Iba1 and Gal-3 were investigated, in relation to macrophages expressing CD68 (ED1; reflecting increased phagocytosis) and CD163 (ED2; implying proinflammatory factor productions) in centrilobular lesions induced in rat livers with thioacetamide (TAA; 300 mg/kg body weight, once intraperitoneally). In agreement with expression patterns of CD68(+) and CD163(+) macrophages, cells reacting to Iba1 and Gal-3 were increased in numbers on post-injection (PI) days 1-5, peaking on day 2; thereafter, the positive cells gradually decreased to control levels until PI days 7 and 10. The increased expressions of Iba1 and Gal-3 were confirmed at mRNA levels by the RT-PCR. Double immunofluorescence staining on PI days 2 and 3 demonstrated Iba1 expression in 15-46% of CD68(+) and CD163(+) macrophages, and Gal-3 expression in 65-82% of CD68(+) and CD163(+) macrophages; Gal-3 expression was observed in 84-93% of Iba1(+) cells. Interestingly, Gal-3 was also expressed in a small number of α-smooth muscle actin-positive myofibroblasts in fibrotic lesions developed in injured centrilobular areas. These findings indicate that macrophages with various functions can participate in development of liver lesions and resultant fibrosis. Besides CD68 and CD163, Iba1 and Gal-3 immunohistochemistry for macrophages would be useful to analyze the pathogenesis behind developing hepatotoxicity.

Changes of serum VEGF concentration after intravitreal injection of Avastin in treatment of diabetic retinopathy

Avastin (bevacizumab) intravitreal injections are widely used for treatment of diabetic retinopathy. The aim of our study was to analyze effect of 1.25 mg of intravitreal Avastin on serum concentration of vascular endothelial growth factor (VEGF) in diabetic patients. Participants were 10 diabetic patients on insulin therapy, without any other eye or systemic disease, and no kidney disfunction. Both eyes of diabetic patients were injected simultaneously with 1.25 mg of intravitreal Avastin, as a first step in treatment of nonproliferative diabetic retinopathy with clinically significant macular edema (4 patients), and of proliferative diabetic retinopathy (6 patients). Fluorescein angiography was performed prior to and laser therapy followed 1 month after Avastin treatment. VEGF concentration in patients serum was measured by ELISA technique: on the day of the Avastin administration, and 1, 7, and 28 days after intravitreal injection. In all analyzed participants, 24 hours after Avastin treatment, serum levels of VEGF were lower then basal (preinjection value). Maximal reduction of serum VEGF was noted on the 7th postoperative day. Twenty-eight days after, VEGF level in serum was raised, without completely reaching basal preoperative concentrations in most patients. Intravitreal injections of anti-VEGF drugs have an effect on decreasing systemic VEGF values. Rhythm of changes in serum VEGF concentrations and lowest detected concentration on the seventh postinjection day are according to pharmacokinetics of Avastin in serum and vitreous, reported by similar studies. The small number of patients involved in this pilot study implicates the need for further studies.

Ocular Pharmacokinetics of Bevacizumab in Vitrectomized Eyes with Silicone Oil Tamponade

We characterized the ocular pharmacokinetics of bevacizumab in vitrectomized eyes with silicone oil tamponade. A total of 18 pigmented rabbits underwent vitrectomy and silicone oil tamponade before an intra-silicone oil injection of 1.25 mg bevacizumab. At post-injection days 1, 7, 14, 28, 42, and 56, 3 rabbits were sacrificed and enucleated, and bevacizumab concentrations were measured in various ocular tissues and plasma. The bevacizumab peak concentration was reached at day 14 in the aqueous humor (4030.70 ng/mL), retina (42,171.7 ng/g), and choroid (56,243.33 ng/g). In the iris/ciliary body and plasma, the peak concentration was reached at day 7 with 52,648.30 ng/g and 197.70 ng/mL, respectively. The choroid had the maximum exposure to bevacizumab with an area under the curve calculated from time zero to the last observed time (AUC(last)) of 1,151,633.40 ng/day/g and the aqueous humor had the minimum exposure (AUC(last) = 74,611.28 ng/day/g) among the ocular tissues, while the drug exposure to the plasma was the smallest of all tissues studied (AUC(last) = 3795.17 ng/day/g). The terminal half-lives and the mean residence time of bevacizumab in the ocular tissues ranged from 3-5 and 10-13 days, respectively. The peak concentration of bevacizumab in various ocular tissues and plasma was delayed and lower than that found in normal rabbit eyes; however, the terminal half-lives were similar to those found in the eyes with native vitreous following an intravitreal injection. Oil may have impacted the distribution of bevacizumab and led to an altered profile of drug level in the ocular tissues.

Leptin Is Involved in Vascularization of Developing Luteal Tissue.

Over half of idiopathic miscarriages are attributed to corpora luteal (CL) defects. Defective luteal tissue typically arises from aberrant luteal development, which commonly results in a decrease in progesterone production. Numerous factors have been identified in the regulation of luteal development including improper vascularization of the developing CL. Several different angiogenic regulators have been identified as playing an important role in vascularization of luteal tissue including vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2), and angiopoietin 1 (Ang-1). All three factors stimulate vascular growth in non-ovarian tissues where they are regulated by various growth promotants including leptin. Leptin is an important metabolic hormone known to have both reproductive and angiogenic properties. Previously, we reported that the expression of both leptin and its receptor is highest in the early stage of the luteal phase and that VEGF, FGF-2, and Ang-1 expression are upregulated by leptin in early, developing luteal tissue. Furthermore, it was determined that blocking ovarian leptin changed luteal vascular morphology and significantly altered the number of large luteal cells present in a day 15 CL. Collectively, the evidence suggests that leptin may be involved in angiogenesis of developing luteal tissue. Therefore, it is hypothesized that ovarian leptin is involved in the vascularization of the developing CL. Eleven cycling crossbred does of similar age were randomly allocated to one of four treatment groups: Control [C; saline in 2.5U heparin (vehicle) only; n=3], rabbit anti-leptin antibody + saline [AL+S; AL antibody (1:10 dilution) in vehicle + vehicle only; n=3], saline + leptin [S+L; vehicle + leptin (1ug, ovine leptin) in vehicle; n=2), and rabbit anti-leptin antibody + leptin (AL+L; in vehicle; n=3). Females were checked twice daily for classical, behavioral estrus and on day 10 of the 3rd estrous cycle does were synchronized (PGF2a administration) for the surgical insertion of a 7-day osmotic infusion pump-catheter apparatus, in apposition to each ovarian hilus, 48 hr post injection (day 0; estrus). Catheter and pump apparatus infused the first treatment (vehicle, anti-leptin, or leptin) over a period of 3 days followed by the second treatment (vehicle, anti-leptin, or leptin) over a period of 4 days, at a flow rate of 0.5 μl/hr. On day 8, ovaries were surgically removed for CL collection and analysis. Gross morphology of CL tissue was recorded and either snap frozen in liquid nitrogen for mRNA analysis or paraffin embedded for microscopic evaluation. Blood samples were collected via jugular venipuncture, from day 0 until day 8 for analysis of serum progesterone during the infusion period. Data was analyzed using MIXED procedures of SAS. Luteal tissue from the S+L and AL+L treatment was underdeveloped but highly vascular. Moreover, progesterone production was low (P<0.05) in all treatment groups compared to the control group. Although luteal expression of VEGF and FGF2 did not differ, Ang-1 was significantly lower (P<0.05) in all treatments compared to the control group. Collectively, the evidence supports the supposition that leptin appears to be involved in vascularization of the developing CL. This research was funded by NIH-NIGMS-MBRS Award #1SCGM095443-01.

Bone marrow mesenchymal stem cells attenuate lung inflammation of hyperoxic newborn rats

BPD is a significant global health problem and currently lacks effective therapy. We established a neonatal rat model of BPD to investigate therapeutic potential of BMSCs in neonatal lung disease. BMSCs were isolated, identified, and transfected by lentiviral vector carrying GFP gene in vitro. Neonatal rats were injected intravenously with either BMSCs or PBS after they had been already exposed to high oxygen for seven days, and assessed on post-injection day 3, day 7, and day 14 for weight gaining, lung histology, radical alveolar counts, and lung cytokine level. BMSCs were positive for CD29, CD44, and CD90 whereas negative for CD34, CD45, CD11b and with differentiation potential into osteoblasts, adipocytes, and chondrocytes. BMSCs expressed GFP after transfected by lentivirus. After injection, BMSCs exert their therapeutic benefit of improving weight gaining, preventing alveolar growth arrest, and suppressing lung inflammation of neonatal rats. Intravenous delivery of BMSCs in newborn rats conferred protection from hyperoxia-induced lung injury, and one of the effects of BMSCs treatment is suppressing lung inflammation.

Forced-exercise attenuates experimental autoimmune neuritis

Physical inactivity in combination with a sedentary lifestyle is strongly associated with an increased risk of development of inflammatory-mediated diseases, including autoimmune disorders. Recent studies suggest that anti-inflammatory effects of physical exercise may be of therapeutic value in some affected individuals. In this study, we determined the effects of forced-exercise (treadmill running) on the development and progression of experimental autoimmune neuritis (EAN), an established animal model of Guillain–Barré syndrome. Adult male Lewis rats were subjected to sedentary (control) or forced-exercise (1.2km per day, 5days a week) for three weeks prior to induction of EAN. P2 (53–78)-immunized sedentary control rats developed a monophasic course of EAN beginning on post-injection day 12.33±0.59 (n=18) and reaching peak severity on day 15.83±0.35 (n=18). At near peak of disease, ankle- and sciatic notch-evoked compound muscle action potential (CMAP) amplitudes in sedentary control rats were reduced (∼50%) while motor nerve conduction velocity (MNCV) was slowed (∼30%) compared with pre-induction evoked responses. In marked contrast, rats undergoing forced-exercise exhibited a significantly less severe clinical course of EAN beginning on post-injection day 12.63±0.53 (n=16) and reaching peaking severity on day 14.69±0.73 (n=16). At near peak of disease, ankle- and sciatic-notch-evoked CMAP amplitudes in forced-exercised rats were preserved while EAN-associated slowing of MNCV was modestly attenuated by exercise. Three weeks of forced-exercise reduced by 46% total plasma corticosterone content while elevating the levels of corticosteroid binding globulin. We conclude from this study that forced-exercise administered prior to and during development of EAN affords a novel measure of protection against autoimmune-associated deficits in peripheral nerve evoked responses independent of steroid-induced immune suppression.

Effect of 2G8, a TGF-beta-R2 inhibitor, on TGF-beta signaling and migration in an immunocompetent pancreatic cancer model.

230 Background: TGF-beta functions as a tumor suppressor early in the development of pancreatic ductal adenocarcinoma (PDAC) but during tumor progression after undefined molecular changes TGF-beta switches to a promoter of metastasis, tumor angiogenesis and epithelial to mesenchymal transition (EMT.) A potential strategy is to target TGF-beta-R2, one of the major signaling receptors for TGF-beta. 2G8 is a anti-rat monoclonal antibody that binds specifically to and blocks murine TGF-beta-R2. Methods: Protein expression was analyzed using Western Blot. Cell proliferation was analyzed using Cell Titer Blue Assay and migration was determined using transwell migration assay. EMT protein expression was determined by ICC. PanO2-HY cells were used to establish an orthotopic and splenic metastasis model. In an orthotopic model, mice received saline, gemcitabine (Gem), 2G8 or 2G8 + Gem. For splenic metastasis model,mice received 2G8 on post injection day −1, +1 or +7 days or control IgG. Results: At baseline, PanO2-HY cells express TGF-beta-R2, alk5 and alk1 and express high levels of zeb1 and beta-catenin. 2G8 inhibits phosphorylation of smad2, ERK1/2 and p38 but not smad1/5 in vitro. 2G8 did not inhibit cell proliferation but did inhibit cell migration in vitro. After 7 days of treatment with 2G8, PanO2-HY cells had reduction in nuclear zeb1 staining by ICC. In the orthotopic model, 2G8 significantly inhibited metastasis to liver, peritoneum and gastrointestinal tract(p=0.02). In splenic metastasis assay, 2G8 treated mice livers weighed significantly less than control treated mice (p=0.0005). In the group of mice treated prior to tumor cell injection, none of them developed a primary tumor. Conclusions: Inhibition of TGF-beta-R2 with 2G8, results in inhibition of smad2 dependent pathways as well as non-canonical TGF-beta signaling in vitro. By inhibiting tumor cell migration in vitro and reducing EMT transcription factors, 2G8 may result in delayed tumor progression. In vivo data in both orthotopic and metastatic models suggest that inhibition of TGF-beta signaling via TGF-beta-R2 inhibits tumor cell metastasis. Further analysis are being conducted to understand changes within primary tumor and metastasis within this model.

Clinical changes in sodium monoiodoacetate–induced stifle osteoarthritis model in dogs

In six dogs, experimental model of osteoarthritis (OA) was reproduced by intraarticular injection of sodium monoiodoacetate (MIA) in left stifle joints. Contralateral joints served as control. The clinical status and some goniometric parameters were monitored before MIA introduction and at post injection days 30, 60 and 105. The results showed convincingly that the used experimental chemical OA model reproduced successfully the disease in canine stifle joints. The studied clinical indices correlated with the severity of disease. ey words: dogs, stifle joint, sodium monoiodoacetate, osteoarthritis

Oral rosmarinic acid‐enhanced Mentha spicata modulates synovial fluid biomarkers of inflammation in horses challenged with intra‐articular LPS

A biological extract of high-rosmarinic acid mint (HRAM) has previously demonstrated inhibitory effects on lipopolysaccharide (LPS)-induced prostaglandin E(2) (PGE(2)), nitric oxide (NO) and glycosaminoglycan (GAG) release in vitro. This study was undertaken to determine whether HRAM added to feed produces similar effects in horses challenged with intra-articular LPS. Eight horses received HRAM (0 or 28.1 ± 1.3 g/day; n = 4 per group) in their feed for 24 days in a blinded manner. On day 21, all horses received an intra-articular injection of LPS (0.3 ng) into their left or right intercarpal joint. Synovial fluid (SF) samples were taken on postinjection day (PID)-21 (i.e. prior to commencement of supplementation), PID0, PID0.25, PID0.5, PID1 and PID3 and analysed for PGE(2), GAG, NO, protein and total nucleated cells counts. Blood biochemistry and haematology screens were conducted at PID-21, PID0, PID1 and PID3. There was a significant reduction in LPS-induced PGE(2) and GAG in SF in horses supplemented with HRAM compared with controls and a tendency to increase complement recognition protein accumulation in synovial fluid of HRAM horses. Plasma from HRAM horses had reduced total white blood cells, segmented neutrophils (compared with baseline concentrations) and lymphocytes (compared with controls), and increased SF nucleated cell count (compared with baseline concentrations and controls). It is concluded that HRAM offered as part of the feed alter biomarkers of inflammation in SF of LPS-challenged horses. Larger studies that seek to clarify effects of HRAM on synovial fluid cell counts and possible role of HRAM-induced interference with complement signalling are warranted.