Post-injection Day Research Articles

Intratendinous Injection of Hyaluronate Induces Acute Inflammation: A Possible Detrimental Effect.

Hyaluronate (HA) is therapeutic for tendinopathy, but an intratendinous HA injection is usually painful; thus, it is not suggested for clinical practice. However, there are no studies on the histopathological changes after an intratendinous HA injection. We hypothesized that an HA injection would induce more-acute inflammation than that induced by an injection of phosphate buffered saline (PBS). Thirty-two rats were randomly divided into 4 post-injection groups (n = 8): day 3, day 7, day 28, and day 42. HA (0.1 c.c.) was, using ultrasound guidance, intratendinously injected into each left Achilles tendon, and PBS (0.1 c.c.) into each right one. For each group, both Achilles tendons of 3 control-group rats (n = 6) were given only needle punctures. The histopathological score, ED1+ and ED2+ macrophage densities, interleukin (IL)-1β expression, and the extent of neovascularization were evaluated. In both experimental groups, each Achilles tendon showed significant histopathological changes and inflammation compatible with acute tendon injury until day 42. The HA group showed more-significant (p < 0.05) histopathological changes, higher ED1+ and ED2+ macrophage density, and higher IL-1β expression than did the PBS group. The neovascularization area was also significantly (p < 0.05) greater in the HA group, except on day 3. Both HA and PBS induced acute tendon injury and inflammation, sequential histopathological changes, ED1+ and ED2+ macrophage accumulation, IL-1β expression, and neovascularization until post-injection day 42.HA induced more-severe injury than did PBS. Therefore, an intratendinous HA injection should be avoided.

Monocular retinal degeneration induced by intravitreal injection of sodium iodate in rabbit eyes.

Our purpose was to evaluate the anatomical and functional changes in retinae of rabbit eyes following monocular intravitreal injection of sodium iodate (SI). Twenty albino rabbits were divided into four groups and underwent monocular intravitreal injection with four different doses of SI (0.1, 0.2, 0.4, and 0.8mg). Before and for 28days after injection, the eyes were examined using fundus photography, optical coherence tomography (OCT), and electroretinography (ERG). At postinjection days 2, 7, and 28, the eyes were enucleated and underwent histological examination. On fundus examination, no distinct retinal changes were seen in any group except the 0.8-mg group, which showed chorioretinal vascular attenuation. In 0.1 and 0.2-mg groups, no significant anatomical changes were found except transient hyperreflective dots over the vitreoretinal interface on OCT. In 0.4 and 0.8-mg groups, disruption of the ellipsoid zone and diffuse retinal swelling were observed in the early period on OCT. In the 0.4-mg group, the outer retina was significantly destroyed at day 28, whereas the inner retina was relatively preserved. In the 0.8-mg group, the entire retina was destroyed irreversibly. The b-wave of ERG was reduced immediately in all groups, which recovered fully (0.1- and 0.2-mg groups), partially (0.4-mg group), or never (0.8-mg group). No structural or functional abnormalities were found in the fellow control eyes. Retinal degeneration following intravitreal injection of SI appears to be dose dependent; retinal damage is reversible at low doses but irreversible at high doses. At a certain dose, the outer retina may be preferably ablated.

Blockade of the spinal BDNF-activated JNK pathway prevents the development of antiretroviral-induced neuropathic pain

Although antiretroviral agents have been used successfully in suppressing viral production, they have also been associated with a number of side effects. The antiretroviral toxic neuropathy induces debilitating and extremely difficult to treat pain syndromes that often lead to discontinuation of antiretroviral therapy. Due to the critical need for the identification of novel therapeutic targets to improve antiretroviral neuropathic pain management, we investigated the role of the JNK signalling pathway in the mechanism of antiretroviral painful neuropathy. Mice were exposed to zalcitabine (2′,3′-dideoxycytidine, ddC) and stavudine (2′,3′-didehydro-3′-deoxythymidine, d4T) that induced a persistent mechanical allodynia and a transient cold allodynia. Treatment with the JNK blocker SP600125 before antiretroviral administration abolished mechanical hypersensitivity with no effect on thermal response. A robust spinal JNK overphosphorylation was observed on post-injection day 1 and 3, along with a JNK-dependent increase in p-c-Jun and ATF3 protein levels. Co-immunoprecipitation experiments showed the presence of a heterodimeric complex between ATF3 and c-Jun indicating that these transcription factors can act together to regulate transcription through heterodimerization. A rise in BDNF and caspase-3 protein levels was detected on day 1 and BDNF sequestration prevented both caspase-3 and p-JNK increase. These data suggest that BDNF plays a role in the early stages of ddC-induced allodynia by promoting apoptotic events and the activation of a hypernociceptive JNK-mediated pathway. We illustrated the activation of a BDNF-mediated JNK pathway involved in the early events responsible for the promotion of neuropathic pain, leading to a better knowledge of the mechanisms involved in the antiretroviral neuropathy. SummaryJNK blockade prevents antiretroviral-induced pain hypersensitivity. This may represent a potential prophylactic treatment of neuropathic pain to improve antiretroviral tolerability.

The early effects of intravitreal anti vascular endothelial growth factor agents on intraocular pressure and central corneal thickness.

To investigate the early effects of two intravitreal (IV) anti vascular endothelial growth factor agents (anti-VEGF), bevacizumab and ranibizumab, on intraocular pressure (IOP) and central corneal thickness (CCT) within the first post-injection month. This prospective study comprised 109 eyes of 109 adult cases who had IV bevacizumab or ranibizumab injections because of age-related macular degeneration (ARMD), retinal venous occlusion (RVO), diabetic retinopathy, and macular edema or central serous chorioretinopathy (CSCR). None of the cases had medical histories of any kinds of glaucoma or increased IOP and IV injection before and all of them underwent a detailed ocular examination including measurements of IOP by non-contact tonometer and CCT by ultrasonic pachymeter pre-injection. IOP measurements were repeated at 30min and 1st, 7th, and 30th day after the injection. CCT measurements were repeated at the 7th and 30th post-injectionday. Paired sample t tests were used for the statistical analysis in order to evaluate the significance of changes in IOP and CCT. The mean age of 56 male and 53 female cases was 63.58±11.04years. Fifty-six cases (51.4%) had diabetic retinopathy, 33 cases (30.3%) had ARMD, 11 cases (10.1%) had RVO, and 9 cases (8.3%) had CSCR. Bevacizumab was used in 97 (89%) cases and ranibizumab was used in 12 (11%) cases. The IOP increased significantly 30min after the injection (p<0.001) but significant decreases were observed at the 1st, 7th, and 30th day post-injection (p<0.001). No significant differences were observed in CCT between pre-injection and 7th and 30th post-injection day values (p=0.924 and p=0.589, respectively). Intravitreal bevacizumab and ranibizumab injections can cause hyper acute increase in IOP because of vitreal expansion but this effect is generally reversible in non-glaucomatous cases.

Immunophenotypical characterization and influence on liver homeostasis of depleting and repopulating hepatic macrophages in rats injected with clodronate

Hepatic macrophages (including Kupffer cells) play a crucial role in the homeostasis and act as mediators of inflammatory response in the liver. Hepatic macrophages were depleted in male F344 rats by a single intravenous injection of liposomal clodronate (CLD; 50mg/kg body weight), and immunophenotypical characteristics of depleting and repopulating macrophages were analyzed by different antibodies specific for macrophages. CD163+ Kupffer cells were almost completely depleted on post-injection (PI) days 1–12. Macrophages reacting to CD68, Iba-1, and Gal-3 were drastically reduced in number on PI day 1 and then recovered gradually until PI day 12. MHC class II+ and CD204+ macrophages were moderately decreased during the observation period. Although hepatic macrophages detectable by different antibodies were reduced in varying degrees, Kupffer cells were the most susceptible to CLD. Liver situation influenced by depleted hepatic macrophages was also investigated. No marked histological changes were seen in the liver, but the proliferating activity of hepatocytes was significantly increased, supported by changes of gene profiles relating to cell proliferation on microarray analysis on PI day 1; the values of AST and ALT were significantly elevated; macrophage induction/activation factors (such as MCP-1, CSF-1, IL-6 and IL-4) were increased exclusively on PI day 1, whereas anti-inflammatory factors such as IL-10 and TGF-β1 remained significantly decreased after macrophage depletion. The present study confirmed importance of hepatic macrophages in liver homeostasis. The condition of hepatic macrophages should be taken into consideration when chemicals capable of inhibiting macrophage functions are evaluated.

Trans-Corneal Subretinal Injection in Mice and Its Effect on the Function and Morphology of the Retina.

PurposeTo introduce a practical method of subretinal injection in mice and evaluate injection-induced retinal detachment (RD) and damage using a dynamic imaging system, electrophysiology, and histology.MethodsAfter full dilation of a 2-month-old C57BL/6J mouse pupil, the cornea near the limbus was punctured with a 30 ½-gague disposable beveled needle. A 33 ½-gauge blunt needle was inserted through the corneal perforation into the anterior chamber, avoiding the lens before going deeper into the vitreous cavity, and penetrating the inner retina to reach the subretinal space. The mice were divided into four groups: in group 1, about 80–100% of the retina was filled with subretinally injected solution; in group 2, approximately 50–70% of the retina was filled with injected solution; in group 3, the procedures were stopped before solution injection; and non-injected eyes were used as the negative control in group 4. An optical coherence tomography (OCT) imaging system was used to monitor retinal reattachment during the first three days following the injections. Histological and functional changes were examined by light microscopy and electroretinography (ERG) at five weeks post-injection.ResultsAfter a short-term training, a 70% success rate with 50% or more coverage (i.e., retinal blebs occupied 50% or more retinal area and filled with the injected solution) with minimal injection-related damages can be achieved. Bleb formation was associated with retinal detachment (RD) between the neuroretina and the retinal pigment epithelium (RPE) layer. Partial RD could be observed at post-injection day 1, and by day 2 most of the retina had reattached. At 5 weeks post-injection, compared to uninjected control group 4, the b-wave amplitudes of ERG decreased 22% in group 1, 16% in group 2, and 7% in group 3; the b-wave amplitudes were statistically different between the uninjected group and the groups with either 50–70% or 80–100% coverage. The subretinal injection-induced RD reattached and became stable at five weeks post-injection, although some photoreceptor damage could still be observed in and around the injection sites, especially in 80–100% coverage group.ConclusionsTrans-corneal subretinal injection is effective and practical, although subretinal injection-related damages can cause some morphological and functional loss.

MRI study of atherosclerotic plaque progression using ultrasmall superparamagnetic iron oxide in Watanabe heritable hyperlipidemic rabbits.

The purpose of this study was to evaluate plaque progression by using MRI with ultrasmall superparamagnetic iron oxide (USPIO) and by histopathological studies. We divided 12 Watanabe heritable hyperlipidemic (WHHL) rabbits into 4 groups based on their age (3, 9, 14 and 26 months) and injected them intravenously with 0.8 mmol (Fe) kg(-1) of USPIO (size, 32 nm; concentration, 15 mg dl(-1)). On the fifth post-injection day, they were again given an intravenous injection with 40 μmol kg(-1) of the same USPIO, and MR angiography (MRA) was performed. The signal-to-noise ratio (SNR) in regions of interest in the wall of the upper abdominal aorta was calculated on coronal images. Specimens from the same level of the aorta were subjected to iron staining and RAM-11 immunostaining and used for histopathological study. For statistical analysis of the MRA and histopathological findings, we used analysis of variance [Tukey's honest significant difference (HSD) test]. In 9-month-old rabbits, the SNR was significantly lower than in rabbits of the other ages (p < 0.01), and the area of RAM-11 (DAKO Corporation, Glostrup, Denmark) and iron uptake in the aortic wall was significantly larger (RAM-11, p < 0.01; iron, p < 0.05). These areas were the smallest in 3-month-old rabbits. Histopathologically, the number of macrophages was the greatest in 9-month-old rabbits. Our findings indicate that the SNR on MRI scans reflects the number of macrophages in the aortic wall of WHHL rabbits. USPIO-enhanced MRI visualized the accumulation of macrophages in early atherosclerotic plaques of WHHL rabbits in the course of natural progression.

Primary Myofibroblasts Maintain Short-Term Viability following Submucosal Injection in Syngeneic, Immune-Competent Mice Utilizing Murine Colonoscopy.

The myofibroblast is an important stromal cell of the gastrointestinal tract. Current in vitro and in vivo models either do not accurately recreate stromal-epithelial interactions or are not specific to myofibroblasts. We sought to create an animal model that would allow the study of myofibroblast-epithelial interactions. We isolated and cultured colonic myofibroblasts from FVB mice. Cells were α-SMA and vimentin positive but desmin negative on immunoblot analysis. We injected the myofibroblasts into the colonic submucosa of syngeneic adult mice (n = 8) via a miniendoscopic system. We then isolated green fluorescent protein (GFP) positive colonic myofibroblasts from C57BL/6-Tg(CAG-EGFP)1Osb/J mice and injected them into the colonic lamina propria of C57BL/6J mice at 1x105 (n = 14), 1x106 (n = 9), or 5x106 cells/mL (n = 4). A subset of mice were injected with serum-free media and ink without cells (n = 3). Mice underwent repeat endoscopy and euthanasia one or 7 days after injection. Colons were isolated and either fixed in 10% formalin or the inked sites were individually excised and lysed for DNA. We assessed the injection sites via histology and immunohistochemical stains for α-SMA and GFP. We used qPCR to quantify GFP DNA transcripts at the lamina propria injection sites. Submucosal injection of myofibroblasts resulted in the formation of a subepithelial wheal on endoscopy, which persisted to day 7. Myofibroblasts injected either into the submucosa or lamina propria maintained viability on post-injection day 7 as evidenced by positive α-SMA staining. qPCR of lamina propria injections showed a dose-dependent increase in GFP DNA transcripts on post-injection day 1, whereas the number of transcripts on day 7 was equivalent for the concentrations injected. We demonstrate short-term survival of primary cultured colonic myofibroblasts in syngeneic mice. This may prove to be a valuable model for studying the role of myofibroblasts in states of health and disease.

Dexamethasone as adjuvant to bupivacaine prolongs the duration of thermal antinociception and prevents bupivacaine-induced rebound hyperalgesia via regional mechanism in a mouse sciatic nerve block model.

BackgroundDexamethasone has been studied as an effective adjuvant to prolong the analgesia duration of local anesthetics in peripheral nerve block. However, the route of action for dexamethasone and its potential neurotoxicity are still unclear.MethodsA mouse sciatic nerve block model was used. The sciatic nerve was injected with 60ul of combinations of various medications, including dexamethasone and/or bupivacaine. Neurobehavioral changes were observed for 2 days prior to injection, and then continuously for up to 7 days after injection. In addition, the sciatic nerves were harvested at either 2 days or 7 days after injection. Toluidine blue dyeing and immunohistochemistry test were performed to study the short-term and long-term histopathological changes of the sciatic nerves. There were six study groups: normal saline control, bupivacaine (10mg/kg) only, dexamethasone (0.5mg/kg) only, bupivacaine (10mg/kg) combined with low-dose (0.14mg/kg) dexamethasone, bupivacaine (10mg/kg) combined with high-dose (0.5mg/kg) dexamethasone, and bupivacaine (10mg/kg) combined with intramuscular dexamethasone (0.5mg/kg).ResultsHigh-dose perineural dexamethasone, but not systemic dexamethasone, combined with bupivacaine prolonged the duration of both sensory and motor block of mouse sciatic nerve. There was no significant difference on the onset time of the sciatic nerve block. There was “rebound hyperalgesia” to thermal stimulus after the resolution of plain bupivacaine sciatic nerve block. Interestingly, both low and high dose perineural dexamethasone prevented bupivacaine-induced hyperalgesia. There was an early phase of axon degeneration and Schwann cell response as represented by S-100 expression as well as the percentage of demyelinated axon and nucleus in the plain bupivacaine group compared with the bupivacaine plus dexamethasone groups on post-injection day 2, which resolved on post-injection day 7. Furthermore, we demonstrated that perineural dexamethasone, but not systemic dexamethasone, could prevent axon degeneration and demyelination. There was no significant caspase-dependent apoptosis process in the mouse sciatic nerve among all study groups during our study period.ConclusionsPerineural, not systemic, dexamethasone added to a clinical concentration of bupivacaine may not only prolong the duration of sensory and motor blockade of sciatic nerve, but also prevent the bupivacaine-induced reversible neurotoxicity and short-term “rebound hyperalgesia” after the resolution of nerve block.

Deferoxamine attenuates lipopolysaccharide-induced neuroinflammation and memory impairment in mice.

BackgroundNeuroinflammation often results in enduring cognitive impairment and is a risk factor for postoperative cognitive dysfunction. There are currently no effective treatments for infection-induced cognitive impairment. Previous studies have shown that the iron chelator deferoxamine (DFO) can increase the resistance of neurons to injury and disease by stimulating adaptive cellular stress responses. However, the impact of DFO on the cognitive sequelae of neuroinflammation is unknown.MethodsA mouse model of lipopolysaccharide (LPS)-induced cognitive impairment was established to evaluate the neuroprotective effects of DFO against LPS-induced memory deficits and neuroinflammation. Adult C57BL/6 mice were treated with 0.5 μg of DFO 3 days prior to intracerebroventricular microinjection of 2 μg of LPS. Cognitive function was assessed using a Morris water maze from post-injection days 1 to 3. Animal behavioral tests, as well as pathological and biochemical assays were performed to evaluate the LPS-induced hippocampal damage and the neuroprotective effect of DFO.ResultsTreatment of mice with LPS resulted in deficits in cognitive performance in the Morris water maze without changing locomotor activity, which were ameliorated by pretreatment with DFO. DFO prevented LPS-induced microglial activation and elevations of IL-1β and TNF-α levels in the hippocampus. Moreover, DFO attenuated elevated expression of caspase-3, modulated GSK3β activity, and prevented LPS-induced increases of MDA and SOD levels in the hippocampus. DFO also significantly blocked LPS-induced iron accumulation and altered expression of proteins related to iron metabolism in the hippocampus.ConclusionsOur results suggest that DFO may possess a neuroprotective effect against LPS-induced neuroinflammation and cognitive deficits via mechanisms involving maintenance of less brain iron, prevention of neuroinflammation, and alleviation of oxidative stress and apoptosis.

IT-19 * TEMOZOLAMIDE RESISTANT INNATE LYMPHOCYTES ADMINISTERED DURING CHEMOTHERAPY IMPROVE SURVIVAL IN HIGH-GRADE GLIOMA

Conventional treatment strategies for high-grade primary brain tumors have failed to consistently extend median survival beyond two years. Using a human/mouse xenograft model, we report improved survival using a combination of Temozolomide (TMZ) chemotherapy with a Drug Resistant Cellular Immunotherapy (DRI) consisting of activated MGMT-transduced γδ T cells and NK cells. MGMT genetic engineering enables cytotoxic lymphocyte function in a chemotherapy-rich environment at a time when the tumor is maximally stressed. Intracranial (IC) glioma xenografts were established using either unmodified (P) or a TMZ-resistant clone (T) of human GBM-X12 primary explants propagated in mice. Tumor-bearing mice were treated i.p. with 60mg/kg TMZ on days 6, 8,13, and 15 post-tumor placement and received IC injection of 1 x 106 MGMT-modified DRI on post-injection days 7 and 14. Control mice received non-modified cells, TMZ alone or no therapy. Survival was assessed using Kaplan-Meier analysis. Both GBM-X12P and X12T express stress antigens MIC-A, MIC-B, and ULBP-4 with only ULBP-4 slightly upregulated on X12T upon exposure to TMZ. Both tumors were killed by expanded/activated γδ T cells in vitro at approximately 80% at an effector : target ratio of 20:1. Cell therapy alone did not improve survival beyond that of untreated mice for either tumor. For the unmodified tumor X12P, both TMZ therapy and TMZ + DRI significantly improved survival over untreated controls (p < 0.001), and the combined therapy increased median survival from 57 to 75 days over TMZ alone. Combined therapy with TMZ + DRI also improved survival for X12T (p = 0.0147) over untreated controls, and showed a marginal improvement over TMZ alone (p = 0.0966) and improvement in median survival from 22 to 25 days. In summary, the combination of chemotherapy-induced stress antigen expression and targeted DRI significantly increases time to progression and improves survival in tumor-bearing immunodeficient mice.

Expression of Nestin in Remodelling of α-Naphthylisothiocyanate-induced Acute Bile Duct Injury in Rats

The function of the intermediate filament protein nestin is poorly understood. The significance of nestin expression was assessed in α-naphthylisothiocyanate (ANIT)-induced cholangiocyte injury lesions in F344 rats. Liver samples obtained from rats injected intraperitoneally with ANIT (75 mg/kg) on post-injection days 0 (control) and 1-12 were labelled immunohistochemically for expression of nestin and markers specific for mesenchymal cells (vimentin), hepatic stellate cells (HSCs) (desmin and glial fibrillary acidic protein [GFAP]), endothelial cells (rat endothelial cell antigen [RECA]-1), cholangiocytes (cytokeratin [CK] 19) and cellular proliferation (Ki67). Cholangiocyte injury led to infiltration of neutrophils and macrophages followed by aggregation of mesenchymal cells and regeneration of bile ducts. Nestin expression was detected in mesenchymal cells (vimentin positive) on days 1-7 with a peak on days 3-5 and in newly-formed RECA-1-positive endothelial cells on day 3. Nestin expression was also observed in regenerating CK19-positive cholangiocytes on days 2-5, with a peak on day 3. Labelling for Ki67 showed proliferation of cholangiocytes, mesenchymal cells and HSCs. Real-time reverse transcriptase polymerase chain reaction with microdissected samples showed significantly elevated nestin mRNA on day 3. The findings suggest an association between nestin expression and cellular proliferation. Based on these findings, it was considered that nestin-expressing mesenchymal cells, HSCs and endothelial cells may be possible progenitors of repopulating cholangiocytes. Nestin expression may serve as an indicator for cellular remodelling and behaviour of injured and repopulating bile ducts.

Management of hypotony after glaucoma filtering surgery

A 39-year-old male with open angle glaucoma in both eyes visited our clinic. The intraocular pressure (IOP) of both eyes fluctuated between 15 mmHg and 25 mmHg. The best corrected visual acuity (BCVA) was 20/20 in the right eye and 20/100 in the left eye. He underwent trabeculectomy with an adjunctive agent (mitomycin C; concentration, 0.2 mg/mL) smoothly on the right eye. After the removal of releasable sutures, the filtering bleb was prominent over the superior limbus for 3 consecutive clock hours with an IOP of about 4–5 mmHg. The Seidel test result was negative, and the anterior chamber depth was moderate to deep. The BCVA of the right eye decreased to 20/100, and optical coherence tomography revealed macular edema. We injected sodium hyaluronate (Healon, Abbott Medical Optics, Santa Ana, CA, USA) into the anterior chamber 2 months after the operation, and repeated the same procedure 4 days later. After each injection, the IOP spiked up to 50 mmHg, and an intravenous infusion of mannitol was required. Sodium hyaluronate was found migrating into the bleb on post-injection day 1. Then we decided to revise the bleb by suturing the scleral flap at two sides. The IOP of the right eye returned to 10 mmHg 4 days after the revision surgery. The BCVA of the right eye recovered to 20/20 6 months after the revision. Optical coherence tomography also showed recovery from macular edema. One IOP-lowering agent (1% brinzolamide ophthalmic suspension) was required for IOP control after bleb revision. Early revision of the scleral flap may resolve hypotony and associated serious complications.

Effect of hyperbaric oxygen therapy on a murine squamous cell carcinoma model

Hyperbaric oxygen (HBO) therapy is sometimes used to assist in wound healing after major head and neck cancer surgery. However, there is concern that HBO treatments might enhance the growth of any residual microscopic disease. This was studied in a mouse model of squamous cell carcinoma (SCC). SCC-VII/SF tumor cells were cultured, and then injected (3 × 10(3) cells) into C3H/HeJ mice in 5 groups: subcutaneous (SQ) control (n = 13), SQ-immediate (n = 12), SQ-delayed (n = 13), tail vein control (n = 8), and tail vein immediate (n = 9). The 3 experimental groups were subjected to HBO therapy, 2.4 atm for 90 minutes, 5 days per week for 4 weeks, starting on postinjection day 3 ("immediate") or 10 ("delayed"). Tumors in the SQ mice were measured 3 times per week. Lung metastases in the tail vein mice were counted at necropsy. At postimplantation day 28, when the immediate group completed its HBO therapy, the tumor volume in the SQ-immediate group was 49.1% higher than the control group, and the SQ-delayed group was 105.1% higher than controls (p < .05). Two weeks later, the SQ-immediate group and SQ-delayed group tumor volumes were still significantly higher than controls, but the difference was smaller (18.4% and 43.8%, respectively; p < .05 only for the delayed group). The tail vein groups had similar numbers of lung metastases, with a mean of 8.7 metastases in the control group and 9.0 metastases in the HBO group (not significant [NS]). This study suggests that HBO therapy does accelerate the growth of microscopic foci of SCCs. This finding differs from some earlier studies and warrants further study.

Establishment of an HBV chronic hepatitis B infection mouse model by vivo transduction of HBV cccDNA

To generate a mouse model of chronic hepatitis B (CHB) infection by performing in vivo transduction of hepatitis B virus (HBV) covalently closed circular (ccc)DNA. Nude mice were injected with HBV cccDNA at doses of 1.5, 1.0 or 0.5 mug/ml. A control group was generated by giving equal injection volumes of physiological saline. The serum levels of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) on post-injection days 1 and 3, weeks 1-6, 8 and 10 were assayed by reflection immunoassay. At post-injection week 10, all animals were sacrificed and liver tissues were collected. Copies of HBV DNA in serum and liver tissue were detected by real-time PCR. HBV antigens in liver tissue were detected of by immunohistochemistry. Pathological analysis of liver tissue carried out with hematoxylin-eosin staining. Linear correlation of data was determined by statistical analysis. HBsAg and HBeAg were detected in sera from all three groups of cccDNA-injected mice staring at post-injection day 1 and lasting through week 10. The levels of HBsAg over the 10-week period showed two patterns of increase-decrease;the lowest level was detected at week 4 and the highest level was detected at week 8. In contrast, the levels of HBeAg over the 10-week period showed three patterns of increase-decrease; the lower levels were detected at weeks 2 and 4 and the higher levels at weeks 3 and 6. HBV DNA copies in liver tissues showed a cccDNA dose-dependent descending trend over the 10-week study period (1.5 mug/ml:1.14E+07 ± 6.51E+06 copies/g, 1.0 mug/ml:9.81E+06 ± 9.32E+06 copies/g, and 0.5 mug/ml:3.72E+06 ± 2.35E+06 copies/g; Pearson's r =0.979). HBV DNA copies in sera showed the pattern of 1.0 mug/ml cccDNA more than 1.5 mug/ml cccDNA more than 0.5 mug/ml cccDNA, and in general were higher than those detected in the liver tissues. Liver tissues from all cccDNA-injected mice showed positive immunohistochemistry staining for both HBsAg and HBeAg. HE staining showed that the liver tissues of all cccDNA-injected mice had severe fatty and vacuolar degeneration and less obvious structure of liver lobules (compared to the liver tissues from control mice). The CHB mouse model successfully established in this study by in vivo transduction of HBV cccDNA may represent a useful tool to study the pathogenic mechanisms and potential antiviral treatments of human CHB.

Blood Glucose Levels in Diabetic Patients Following Corticosteroid Injections Into the Hand and Wrist

To quantify diabetic patients' change in blood glucose levels after corticosteroid injection for common hand diseases and to assess which patient-level risk factors may predict an increase in blood glucose levels. Patients were recruited for this case-crossover study in the clinic of fellowship-trained hand surgeons at a tertiary care center. Patients with diabetes mellitus type 1 or 2, who received a corticosteroid injection, recorded the morning fasting blood glucose levels for 14 days after the injection. Fasting glucose levels on days 1 to 7 after injection qualified as case data; levels on days 10 to 14 provided control data. A mixed model with a priori contrasts was used to compare postinjection blood glucose levels with baseline levels. We used a linear regression model to determine patient predictors of a postinjection rise in blood glucose levels. Of 67 patients recruited for the study returned, 40 (60%) completed blood glucose logs. There was a significant increase in fasting blood glucose levels after injection limited to postinjection days 1 and 2. Among patient risk factors in the linear regression model, type 1 diabetes and use of insulin each predicted a postinjection increase in blood glucose levels from baseline, whereas higher glycated hemoglobin levels did not predict increases. Corticosteroid injections in the hand transiently increase blood glucose levels in diabetic patients. Patients with type 1 diabetes and insulin-dependent diabetics are more likely to experience this transient rise in blood glucose levels. Therapeutic III.

Differential expression in ACE2, Ang(1-7) and Mas receptor during progression of liver fibrosis in a rat model

To investigate the changes in expression of the ACE2/Ang(1-7)/Mas receptor axis' components that occur during progression of liver fibrosis using a rat model system. Thirty-six adult male Wistar rats, were randomly assigned to groups of normal control (n = 6; no manipulation) and liver fibrosis (n = 30; given a subcutaneous injection of 40% chronic carbon tetrachloride (CCl4)). At post-injection days 15, 30, 45, 60 and 75, 1 control rat and 6 modeled rats were sacrificed for analysis. Histopathological analysis of liver tissue was performed with hematoxylin-eosin and rapid Masson staining. Protein expression level of Ang(1-7) was determined by enzyme-linked immunosorbent assay, and of ACE2 and Mas receptor was evaluated by immunohistochemistry. Real-time PCR was used to measure the mRNA expression levels of ACE2 and Mas receptor. The expression levels of ACE2, Ang(1-7) and Mas receptor showed a statistically significant upward trend that followed the progression of fibrosis up to post-injection day 60 (P less than 0.01), but the significant increase was not seen from day 60 to day 75. Each component of the ACE2/Ang(1-7)/Mas receptor axis shows differential expression during the development of liver fibrosis and may contribute to disease progression.

Effect of thiomersalate exposure on the behaviour and humoral immunity of premature rats

Objective To discover the influence of thiomersalate exposure on the behavior and humoral immunity of premature rats. Methods According to common thiomersalate dose of vaccine used in human, the surface conversation method was used to change the dose to 1, 2, 3, 4 times for rats(32.8, 65.6, 98.4 and 131.2 μg/kg), and the thiomersalate was separately injected to the gluteus maximus of premature rats respectively, which were delivered on the 20th day of gestation by cesarean for the injection on day 1(premature). Simultaneously, saline was injected to the control group.On day 44 to 48 after injection, the Morris water maze test was used to evaluate the spatial learning and memory abilities of rats and on the post-injection day 49, immunoglobulin content in rat blood and cerebrospinal fluid was measured. Results The spatial learning abilities of rats in 131.2 μg/kg group and the memory abilities in 65.6 μg/kg, 98.4 μg/kg and 131.2 μg/kg group were significantly weaker than those in 9 g/L saline group(all P<0.001). The immunoglobulin content in blood and cerebrospinal fluid changed in different groups exposed to thiomersalate.The levels of IgG, IgA, IgE, IgM in 98.4 μg/kg group and 131.2 μg/kg group rose significantly higher than those in 9 g/L saline group(all P<0.01). Analysis of rectilinear correlation between the immunoglobulin and learning abilities of rats revealed that IgG, IgA, IgE levels in the blood were correlated with the spatial learning abilities(r=0.36, 0.47, 0.50, all P<0.05)and the levels of IgG, IgA, IgE, IgM in blood were rectilinear correlated with the memory abilities of rats(r=-0.39, -0.43, -0.49, -0.38, all P<0.05). In rats'cerebrospinal fluid, IgG, IgA, IgE, IgM levels were rectilinear correlative with both the spatial learning and memory abilities(r=0.48, 0.59, 0.54, 0.41, all P<0.05; r=-0.39, -0.61, -0.57, -0.44, all P<0.05). Conclusion With the dose of thiomersalate increased, the learning and memory and humoral immunity are sustained damaged for premature rats. Key words: Thiomersalate; Humoral immunity; Premature rat

Tunable sustained intravitreal drug delivery system for daunorubicin using oxidized porous silicon

Daunorubicin (DNR) is an effective inhibitor of an array of proteins involved in neovascularization, including VEGF and PDGF. These growth factors are directly related to retina scar formation in many devastating retinal diseases. Due to the short vitreous half-life and narrow therapeutic window, ocular application of DNR is limited. It has been shown that a porous silicon (pSi) based delivery system can extend DNR vitreous residence from a few days to 3months. In this study we investigated the feasibility of altering the pore size of the silicon particles to regulate the payload release. Modulation of the etching parameters allowed control of the nano-pore size from 15nm to 95nm. In vitro studies showed that degradation of pSiO2 increased with increasing pore size and the degradation of pSiO2 was approximately constant for a given particle type. The degradation of pSiO2 with 43nm pores was significantly greater than the other two particles with smaller pores, judged by observed and normalized mean Si concentration of the dissolution samples (44.2±8.9 vs 25.7±5.6 or 21.2±4.2μg/mL, p<0.0001). In vitro dynamic DNR release revealed that pSiO2–CO2H:DNR (porous silicon dioxide with covalent loading of daunorubicin) with large pores (43nm) yielded a significantly higher DNR level than particles with 15 or 26nm pores (13.5±6.9ng/mL vs. 2.3±1.6ng/mL and 1.1±0.9ng/mL, p<0.0001). After two months of in vitro dynamic release, 54% of the pSiO2–CO2H:DNR particles still remained in the dissolution chamber by weight. In vivo drug release study demonstrated that free DNR in the vitreous at post-injection day 14 was 66.52ng/mL for 95nm pore size pSiO2–CO2H:DNR, 10.76ng/mL for 43nm pSiO2–CO2H:DNR, and only 1.05ng/mL for 15nm pSiO2–CO2H:DNR. Pore expansion from 15nm to 95nm led to a 63 fold increase of DNR release (p<0.0001) and a direct correlation between the pore size and the drug levels in the living eye vitreous was confirmed. The present study demonstrates the feasibility of regulating DNR release from pSiO2 covalently loaded with DNR by engineering the nano-pore size of pSi.

Safety of intravitreal triamcinolone acetonide: an electrophysiologic and histopathological study in rabbits.

To evaluate the retinal safety of various doses of intravitreal triamcinolone acetonide (TA) in rabbits. Thirty New Zealand albino rabbits were divided into five groups (six animals each). In group 1 (control group), each animal received a single intravitreal injection of 0.1mL phosphate buffered saline. In groups 2, 3, 4 and 5, each rabbit received a single intravitreal injection of 4, 8, 16 and 32mg of TA, respectively. Each dose was contained in 0.1mL phosphate buffered saline. Clinical ocular examinations were performed before the injection and on the 1st, 3rd, 10th and 17th post-injection days. A standard dark adapted electroretinogram (ERG) was obtained before injection and on the 3rd, 10th and 17th post-injection days. After 17d, animals were sacrificed and their eyes prepared for pathological examination. By monitoring ERG as a functional index for the retina, intravitreal injection of 4mg TA showed no significant ERG changes. At doses of 8, 16 and 32, hyper-abnormal responses in a- and b- waves of ERG were detected on the 3rd post-injection day. These changes gradually returned back to normal limits after 17d. Histopathological examination of the retina of all animals showed no pathological changes. High doses of intravitreal TA seemed to have enhancing effects on the retinal function with gradual return to normal limits with no pathological changes detected in examined eyes.