Therapeutic effect of exendin-4, a long-acting analogue of glucagon-like peptide-1 receptor agonist, on nerve regeneration after the crush nerve injury.

Glucagon-like peptide-1 (GLP-1) is glucose-dependent insulinotropic hormone secreted from enteroendocrine L cells. Its long-acting analogue, exendin-4, is equipotent to GLP-1 and is used to treat type 2 diabetes mellitus. In addition, exendin-4 has effects on the central and peripheral nervous system. In this study, we administered repeated intraperitoneal (i.p.) injections of exendin-4 to examine whether exendin-4 is able to facilitate the recovery after the crush nerve injury. Exendin-4 injection was started immediately after crush injury and was repeated every day for subsequent 14 days. Rats subjected to sciatic nerve crush exhibited marked functional loss, electrophysiological dysfunction, and atrophy of the tibialis anterior muscle (TA). All these changes, except for the atrophy of TA, were improved significantly by the administration of exendin-4. Functional, electrophysiological, and morphological parameters indicated significant enhancement of nerve regeneration 4 weeks after nerve crush. These results suggest that exendin-4 is feasible for clinical application to treat peripheral nerve injury.

Proteomic analysis of the lung in rats with hypobaric hypoxia-induced pulmonary hypertension.

Experimental pulmonary hypertension that develops in hypobaric hypoxia is characterized by structural remodeling of the lung. Proteomics - which may be the most powerful way to uncover unknown remodeling proteins involved in enhancing cardiovascular performance - was used to study 150 male Wistar rats housed for up to 21 days in a chamber at the equivalent of 5500 m altitude level. After 14 days' exposure to hypobaric hypoxia, pulmonary arterial pressure (PAP) was significantly increased. In lung tissue, about 140 matching protein spots were found among 8 groups (divided according to their hypobaric period) by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) (pH4.5-pH6.5, 30 kDa-100 kDa). In hypobaric rats, three spots were increased two-fold or more (vs. control rats) in two-dimensional differential in-gel electrophoresis (2D-DIGE). The increased proteins were identified, by matrix-assisted laser desorption ionization time of flight (MALDI-TOF), as one isoform of heat shock protein 70 (HSP70) and two isoforms of protein disulfide isomerase associated 3. This result was confirmed by Western blotting analysis of 2D-PAGE. Conceivably, HSP70 and PDIA3 may play roles in modulating the lung structural remodeling that occurs due to pulmonary hypertension in hypobaric hypoxia.

Expression of P2X4R mRNA and protein in rats with hypobaric hypoxia-induced pulmonary hypertension.

BACKGROUND: The experimental pulmonary hypertension that develops in hypobaric hypoxia is characterized by structural remodeling of the heart. The P2X4 receptor (P2X4R) controls vascular tone and vessel remodeling in several blood vessels, and it has emerged as a key factor in the enhancement of cardiovascular performance. METHODS AND RESULTS: To study the possible effects of hypobaric hypoxia on the P2X4R-synthesis system, 150 male Wistar rats were housed in a chamber at the equivalent of the 5,500 m altitude level for 21 days. After 14 days' exposure to hypobaric hypoxia, pulmonary arterial pressure (PAP) was significantly increased. In the right ventricle (RV) of the heart, P2X4R expression was significantly increased on days 1 and 14 (mRNA) and on days 7 and 21 (protein) of hypobaric hypoxic exposure. Immunohistochemical staining for P2X4R protein became more intense in RV in the late phase of exposure. These changes in P2X4R synthesis in RV occurred alongside the increase in PAP. In addition, P2X1R and P2Y2R mRNA levels in the RV were significantly increased on days 1, 14, and 21, and day 5, respectively, of exposure. The level of P2X1R protein in the RV was significantly increased on day 21 of exposure. CONCLUSIONS: Conceivably, P2 receptors, including P2X4R and P2X1R, might play roles in modulating the RV hypertrophy that occurs due to pulmonary hypertension in hypobaric hypoxia.

Axonal-transport-mediated gene transduction in the interior of rat bone.

BACKGROUND: Gene transduction has been considered advantageous for the sustained delivery of proteins to specific target tissues. However, in the case of hard tissues, such as bone, local gene delivery remains problematic owing to anatomical accessibility limitations of the target sites. METHODOLOGY/PRINCIPAL FINDINGS: Here, we evaluated the feasibility of exogenous gene transduction in the interior of bone via axonal transport following intramuscular administration of a nonviral vector. A high expression level of the transduced gene was achieved in the tibia ipsilateral to the injected tibialis anterior muscle, as well as in the ipsilateral sciatic nerve and dorsal root ganglia. In sciatic transection rats, the gene expression level was significantly lowered in bone. CONCLUSIONS/SIGNIFICANCE: These results suggest that axonal transport is critical for gene transduction. Our study may provide a basis for developing therapeutic methods for efficient gene delivery into hard tissues.

Nonviral retrograde gene transfer of human hepatocyte growth factor improves neuropathic pain-related phenomena in rats.

Peripheral nerve injury occasionally causes chronic neuropathic pain with hyperalgesia and allodynia. However, its treatment is difficult. Here, we used a chronic constriction injury (CCI) model in rats to investigate the effects on experimental neuropathic pain of the human hepatocyte growth factor (HGF) gene delivered into the nervous system by retrograde axonal transport following its repeated intramuscular transfer, using liposomes containing the hemagglutinating virus of Japan (HVJ). CCI (control) rats exhibited marked mechanical allodynia and thermal hyperalgesia, and decreased blood flow in sciatic nerve and hind paw. All these changes were significantly reversed by HGF gene transfer. In the sciatic nerve in HGF-treated rats, the size-frequency distributions for myelinated and unmyelinated axons each showed a rightward shift, the number of myelinated axons >5 microm in diameter was significantly increased, and the mean diameter of unmyelinated axons was significantly increased (versus CCI rats). Levels of P2X3, P2X4, and P2Y1 receptor mRNAs, and of interleukin-6 (IL-6) and activating transcription factor 3 (ATF3) mRNAs, were elevated in the ipsilateral dorsal root ganglia and/or sciatic nerve by CCI, and these levels were decreased by HGF gene transfer. These results may point toward a potential new treatment strategy for chronic neuropathic pain in this model.

A novel autonomic activation measurement method for stress monitoring: non-contact measurement of heart rate variability using a compact microwave radar.

We developed a novel method for non-contact monitoring of stress-induced autonomic activation through the back of a chair, using a compact 24 GHz microwave radar (8 x 5 x 3 cm), without large-scale equipment and placing a heavy burden on the monitored individual. Following a silent period of 120 s, audio stimuli using a composite tone of 2,120 and 2,130 Hz sine-waves at 95 dB were conducted for 120 s. From dorsal, LF/HF of HRV reflecting sympatho-vagal balance was determined by microwave radar with the maximum entropy method using eight volunteers (mean age 23 +/- 1 years). Mean LF/HF measured by non-contact and contact (using electrocardiography for reference) methods during audio stimuli increased 34 and 37%, respectively, as compared with those of the silent period. Maximum cross-correlations between contact and non-contact measurements averaged 0.73 +/- 0.10. Our method appears to be promising for future monitoring of stress-induced autonomic activation of operators and may reduce stress-induced accidents.

Non-contact respiratory monitoring system using a ceiling-attached microwave antenna.

Using a microwave antenna attached to the room ceiling, we conducted non-contact monitoring of respiratory chest wall motions of subjects in bed and covered by a soft comfortable bedding, to measure the vital signs of patients under nursing care in a welfare institution. Long-term vital sign monitoring using electrodes places a heavy burden on monitored individuals. Our non-contact respiratory monitoring system comprises a 1,215 MHz-microwave radar (LDR-1), antenna box attached to the ceiling, and personal computer with analyzing software. The system was tested on eight healthy volunteers (mean age, 25 years; range, 21-44 years) and eight elderly volunteers with some disorders (mean age, 69 years; range, 66-75 years). Respiratory rates of subjects measured using this system correlated with rates measured using respiration sensors (r=0.97, P<0.001 for healthy volunteers, r=0.98, P<0.0001 for elderly volunteers). The system could monitor subtle changes in respiratory rate, and monitoring respiratory rate increases caused by disorders such as pneumonia will be possible.

In vitro gene transfer to mammalian cells by the use of laser-induced stress waves: effects of stress wave parameters, ambient temperature, and cell type.

Laser-mediated gene transfection has received much attention as a new method for targeted gene therapy because of the high spatial controllability of laser energy. We previously demonstrated both in vivo and in vitro that plasmid DNA can be transfected by applying nanosecond pulsed laser-induced stress waves (LISWs). In the present study, we investigated the dependence of transfection efficiency on the laser irradiation conditions and hence stress wave conditions in vitro. We measured characteristics of LISWs used for gene transfection. For NIH 3T3 cells, transfection efficiency was evaluated as functions of laser fluence and number of pulses. The effect of ambient temperature was also investigated, and it was found that change in ambient temperature in a specific range resulted in drastic change in transfection efficiency for NIH 3T3 cells. Gene transfection of different types of cell lines were also demonstrated, where cellular heating increased transfection efficiency for nonmalignant cells, while heating decreased transfection efficiency for malignant cells.

Nonviral HVJ (hemagglutinating virus of Japan) liposome-mediated retrograde gene transfer of human hepatocyte growth factor into rat nervous system promotes functional and histological recovery of the crushed nerve.

Hepatocyte growth factor (HGF) is well known to be involved in many biological functions, such as organ regeneration and angiogenesis, and to exert neurotrophic effects on motor, sensory, and parasympathetic neurons. In this study, we gave repeated intramuscular injections of the human HGF gene, using nonviral HVJ (hemagglutinating virus of Japan) liposome method, to examine whether transfection of the rat nervous system with this gene is able to exert neurotrophic effects facilitating recovery of a crushed nerve. The expression of HGF protein and HGF mRNA indicated that gene transfer into the nervous system did occur via retrograde axonal transport. At 4 weeks after crush, electrophysiological examination of the crushed nerve showed a significantly shorter mean latency and a significantly greater mean maximum M-wave amplitude with repeated injections of HGF gene. Furthermore, histological findings showed that the mean diameter of the axons, the axon number and the axon population were significantly larger in the group with repeated injections of HGF gene. The above results show that repeated human HGF gene transfer into the rat nervous system is able to promote crushed-nerve recovery, both electrophysiologically and histologically, and suggest that HGF gene transfer has potential for the treatment of crushed nerve.

Expression of hypoxia-inducible factor-1alpha in esophageal squamous cell carcinoma.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays an important role in tumor growth and metastasis by regulating energy metabolism and inducing angiogenesis. Elevated levels of HIF-1alpha, a subunit of HIF-1, are noted in various malignant tumors, but it is unclear whether this is so in esophageal carcinoma. The purpose of this study was to evaluate the implications of HIF-1alpha expression in esophageal squamous cell carcinoma. In 215 patients with esophageal carcinoma, we examined immunoreactivity for HIF-1alpha protein, vascular endothelial growth factor (VEGF) protein and p53 protein. In 38 patients, we examined the expression of HIF-1alpha messenger ribonucleic acid (mRNA) (using the semiquantitative reverse transcriptase-polymerase chain reaction [RT-PCR]). A positive HIF-1alpha protein expression was recognized in 95% of the patients, and was strongly apparent within both the nuclei and/or cytoplasm of tumor cells. The proportion of patients in the 'high score' group for HIF-1alpha protein expression increased significantly with increasing VEGF protein expression. Immunoreactivity for HIF-1alpha protein was found to have a significant effect on disease-free survival rate in our univariate analysis, but no effect on overall survival rate. In RT-PCR, HIF-1alpha mRNA scores correlated significantly with scores for HIF-1alpha protein expression, but not with any clinicopathologic factor or either of the survival rates. The detection of HIF-1alpha protein and mRNA would appear to offer limited information as to progression and prognosis in esophageal carcinoma.

Nonviral gene transfer of human hepatocyte growth factor improves streptozotocin-induced diabetic neuropathy in rats.

Peripheral neuropathy is common and ultimately accounts for significant morbidity in diabetes. Recently, several neurotrophic factors have been used to prevent progression of diabetic neuropathy. In this study, we gave repeated intramuscular injections of the human hepatocyte growth factor (HGF) gene percutaneously, using liposomes containing the hemagglutinating virus of Japan (HVJ), to examine therapeutic efficacy of nonviral gene transfer of HGF for experimental diabetic sensorimotor neuropathy in rats. Experimental diabetes induced by intraperitoneal injection of streptozotocin resulted in a marked tactile allodynia (but not in a thermal hyperalgesia), in a reduction of both the conduction velocity and the amplitude, and in a decreased laser Doppler flux of the nerve and the muscle at 6 weeks after the induction. All these changes were significantly reversed by repeated gene transfer of HGF. Furthermore, we analyzed the density of endoneurial capillaries and morphometrical changes of the nerve. The density of endoneurial capillaries, disclosing marked reduction in diabetic rats, was also reversed significantly by repeated gene transfer of HGF; however, no considerable differences were observed morphometrically in either myelinated or unmyelinated axons. These results suggest that nonviral HVJ liposome-mediated gene transfer of human HGF has potential for the safe effective treatment of diabetic sensorimotor neuropathy.

Expression of endothelin-1 in the brain and lung of rats exposed to permanent hypobaric hypoxia.

High-altitude hypoxia causes pulmonary hypertension in humans and animals. Endothelin-1 (ET-1) is a novel and long-lasting vasoconstrictor. However, no study has dealt with the effects of a hypobaric hypoxic environment (HHE) on ET-1 activity in the brain. We examined 134 male rats permanently exposed to the equivalent of 5500 m altitude for 1 to 8 weeks. In these HHE rats, the mean pulmonary arterial pressure was significantly raised. The level of ET-1 protein, measured by enzyme immunoassay, increased rapidly in the lungs on exposure to HHE, but decreased in the brain. The level of ET-1 mRNA, measured by semiquantitative RT-PCR, was raised at 1, 4, and 6 weeks' exposure in the lungs and at 4 or more weeks' exposure in 3 of 8 brain regions. By in situ hybridization and immunohistochemistry of brain sections, ET-1 mRNA and protein were detected in the endothelial cells, neurons, and astrocyte-like cells in control rats. In HHE rats, the immunoreactive intensity for ET-1 protein decreased rapidly with time in these cells within the brain, although a few weakly ET-1 protein-positive cells were detected until 8 weeks' exposure to HHE. Only a few weakly ET-1 mRNA-positive endothelial cells were detected in any HHE rats. Although the reactivity for ET-1 mRNA had decreased significantly in neurons and astrocyte-like cells at 1 and 2 weeks' exposure to HHE, it was again strong in both types of cells at 4 weeks' exposure to HHE. These results raise the possibility that during exposure to HHE, ET-1 production in the lung may play a role in the development of pulmonary hypertension, while a decrease in ET-1 production within the brain may help to protect neurons by preventing or limiting the constriction of cerebral microvessels during the hypoxia induced by HHE.

Usefulness of photocrosslinkable chitosan for endoscopic cancer treatment in alimentary tract.

Endoscopic mucosal resection (EMR), an established treatment for superficial cancer in the stomach, colon, and esophagus, involves extracting the lesion with the aid of high-frequency current (as in snare polypectomy), following saline injection into the submucosal layer. Of interest in this study was the ability of photocrosslinkable chitosan, which is converted to a hydrogel (like a soft rubber) by 30-s ultraviolet irradiation, to expand the submucosal layer when used as an injection material. Photocrosslinkable-chitosan solution or normal saline (0.3 mL) was injected into the submucosal layer of the rat stomach, and the thickness of this layer was measured at various time points thereafter. In these two groups, the values obtained were 3.8 and 2.0 mm, respectively (means; n = 5 each group; p < 0.005), at 30 min after the injection. The cumulative blood loss measured at 20 min after mucosal incision was 113 mg (photocrosslinkable-chitosan solution), versus 1682 mg (saline) (means, n = 10 each group; p < 0.001). Histologic observation revealed that chitosan, which was retained within the submucosal layer, filled the ulcer base and completely surrounded the bleeding focus. Thus, the photocrosslinkable chitosan developed holds promise for use in EMR as a submucosal injection agent that avoids complications.

Gene transfer into mammalian cells by use of a nanosecond pulsed laser-induced stress wave.

Plasmid DNA has been successfully delivered to mammalian cells by applying a nanosecond pulsed laser-induced stress wave (LISW). Cells exposed to a LISW were selectively transfected with plasmids coding for green fluorescent protein. It was also shown that transient, mild cellular heating (approximately 43 degrees C) was effective in improving the transfection efficiency.

In vivo targeted gene transfer in skin by the use of laser-induced stress waves.

BACKGROUND AND OBJECTIVES: Much interest has been shown in the use of lasers for nonviral targeted gene transfer, since the spatial characteristics of laser light are quite well defined. The aim of this study was to demonstrate in vivo gene transfer by the use of laser-induced stress waves (LISWs). STUDY DESIGN/MATERIALS AND METHODS: After reporter genes had been intradermally injected to rat skin in vivo, a laser target was placed on the gene-injected skin. LISWs were generated by the irradiation of an elastic laser target with 532-nm nanosecond laser pulses of a Q-switched Nd:YAG laser. RESULTS: Levels of luciferase activities for the skin exposed to LISWs were two orders of magnitude higher than those for the skin injected with naked DNA. Expressions of enhanced green fluorescent protein (EGFP) and beta-galactosidase were observed only in the area that was exposed to LISWs, and in addition, epidermal cells were selectively transfected. No major side effects were observed, and luciferase activity levels as high as 10(5) RLU per mg of protein were sustained even 5 days after gene transfer. CONCLUSION: Highly efficient and site-specific gene transfer can be achieved by applying a few pulses of nanosecond pulsed LISWs to rat skin in vivo.

Expressions of adrenomedullin mRNA and protein in rats with hypobaric hypoxia-induced pulmonary hypertension.

Experimental pulmonary hypertension induced in a hypobaric hypoxic environment (HHE) is characterized by structural remodeling of the heart and pulmonary arteries. Adrenomedullin (AM) has diuretic, natriuretic, and hypotensive effects. To study the possible effects of HHE on the AM synthesis system, 150 male Wistar rats were housed in a chamber at the equivalent of a 5,500-m altitude level for 21 days. After 14 days of exposure to HHE, pulmonary arterial pressure (PAP) was significantly increased (compared with control rats). The plasma AM protein level was significantly increased on day 21 of exposure to HHE. In the right ventricle (RV), right atrium, and left atrium of the heart, the expressions of AM mRNA and protein were increased in the middle to late phase (5-21 days) of HHE, whereas in the brain and lung they were increased much earlier (0.5-5 days). In situ hybridization and immunohistochemistry showed AM mRNA and protein staining to be more intense in the RV in animals in the middle to late phase of HHE exposure than in the controls. During HHE, these changes in AM synthesis, which occurred strongly in the RV, occurred alongside the increase in PAP. Conceivably, AM may play a role in modulating pulmonary hypertension in HHE.

Efficient gene transfer from innervated muscle into rat peripheral and central nervous systems using a non-viral haemagglutinating virus of Japan (HVJ)-liposome method.

We evaluated the feasibility of gene delivery into the peripheral and central nervous systems via retrograde axonal transport following injection of a haemagglutinating virus of Japan (HVJ)-liposome-DNA complex vector into an innervated muscle. Transfection efficiency was assessed by measuring luciferase activity, and was compared statistically with that achieved using a liposome-DNA control vector. High luciferase activity was observed in the injected muscle, the ipsilateral sciatic nerve, and the ipsilateral dorsal root ganglia on day 1 after gene transfer. The spinal cord also showed luciferase activity, although this was lower than in the other tissues. However, no activity was observed in the contralateral sciatic nerve or the contralateral dorsal root ganglia. In addition, we performed gene transfer twice, with a 1-week interval, to evaluate the feasibility of repeated therapeutic gene delivery. Again, a high transfection efficiency was observed immediately, even after the second gene transfer, and transfection efficiency was significantly higher at each defined time-point using the HVJ-liposome complex vector than using a control vector. These results indicate that this method could be used for repeated therapeutic gene delivery into muscle, nerve, dorsal root ganglia, and possibly spinal cord, without the need for a surgical approach, making it well suited to clinical applications.

Gene transfer of human hepatocyte growth factor into rat skin wounds mediated by liposomes coated with the sendai virus (hemagglutinating virus of Japan).

Hepatocyte growth factor (HGF) regulates cell growth, cell motility, and morphogenesis in various types of cells, including epithelial and endothelial cells, indicating that it probably promotes epithelial repair and neovascularization during wound healing. To better understand the effects of HGF on wound healing, we performed human HGF-gene transfer into skin wounds in rats. The rat HGF mRNA levels, and human and rat HGF protein concentrations in the wounds in HGF gene-transfer rats were significantly elevated at 3 days, 3 to 14 days, and 3 and 14 days after gene transfer, respectively. An expression of human HGF mRNA and protein was revealed in squamous cells in the epidermis, in endothelial cells and smooth muscle cells in blood vessels, and in fibroblasts in granulation tissues at 3, 7, and 14 days after gene transfer in HGF gene-transfer rats. The wound lesion area in HGF gene-transfer rats was significantly less than that in control rats from 3 to 7 days after gene transfer. The re-epithelialization rate, microvessel counts in granulation tissues, proliferating cell nuclear antigen index of fibroblasts in granulation tissues, and the proliferating cell nuclear antigen index in the epidermis of HGF gene-transfer rats were significantly increased at 3 and 7 days after gene transfer. Semiquantitative reverse transcriptase-polymerase chain reaction revealed that the expression levels of transforming growth factor-beta1 and Colalpha2(I) mRNAs in the wounds of HGF gene-transfer rats were significantly decreased at 7 and 14 days, respectively. The hydroxyproline concentration in the wound was significantly less in HGF gene-transfer rats than in control rats at 3 days after gene transfer. These results suggest that HGF gene transfer into a skin wound may aid re-epithelialization and neovascularization in the early phase of wound healing, and that HGF may play a role in modulating cutaneous wound healing.

Changes in myosin heavy chain and its localization in rat heart in association with hypobaric hypoxia-induced pulmonary hypertension.

Experimental pulmonary hypertension induced in a hypobaric hypoxic environment (HHE) is characterized by structural remodelling of the heart. In rat cardiac ventricles, pressure and volume overload are well known to be associated with changes in cardiac myosin heavy chain (MHC) isoforms. To study the effects of HHE on the MHC profile in the ventricles, 83 male Wistar rats were housed in a chamber at the equivalent of 5500 m altitude for 1-8 weeks. Pulmonary arterial pressure, right ventricular free wall (RVFW) weight, the ratio of RVFW weight over body weight (BW), the ratio of left ventricular free wall (LVFW) weight over BW, and myocyte diameter in both ventricles showed significant increases after 1 week, 2 weeks, 1 week, 6 weeks, and 4 weeks of HHE, respectively. Semi-quantitative reverse transcriptase-polymerase chain reaction revealed that beta-MHC mRNA expression was increased significantly in both ventricles at 6 and 8 weeks of HHE, whereas alpha-MHC mRNA expression was decreased significantly at 6 and 8 weeks of HHE in the right ventricle (RV) and at 6 weeks of HHE in the left ventricle (LV). The percentage of myosin containing the beta-MHC isoform was increased significantly at 4-8 weeks of HHE in RV and at 6 weeks of HHE in LV. In situ hybridization showed that the area of strong staining for beta-MHC mRNA was increased in both ventricles at 8 weeks of HHE, and showed a decrease from RVFW to cardiac septum, and from cardiac septum to LVFW. These results suggest that HHE has a significant effect on the expression of both MHC mRNA and protein in the heart, particularly in RV. These changes may reflect a role for cardiac MHC in the response to pulmonary hypertension in HHE.

Photocrosslinkable chitosan as a dressing for wound occlusion and accelerator in healing process.

Application of ultraviolet light (UV-) irradiation to a photocrosslinkable chitosan (Az-CH-LA) aqueous solution resulted in an insoluble, flexible hydrogel like soft rubber within 60 s. The chitosan hydrogel could completely stop bleeding from a cut mouse tail within 30 s of UV-irradiation and could firmly adhere two pieces of sliced skins of mouse to each other. In order to evaluate its accelerating effect on wound healing, full thickness-skin incisions were made on the back of mice and subsequently an Az-CH-LA aqueous solution was added into the wound and irradiated with UV light for 90 s. Application of the chitosan hydrogel significantly induced wound contraction and accelerated wound closure and healing. Histological examinations also have demonstrated an advanced granulation tissue formation and epithelialization in the chitosan hydrogel treated wounds. The chitosan hydrogel due to its accelerating healing ability is considered to become an excellent dressing for wound occlusion and tissue adhesive in urgent hemostasis situations.