Jiin-Tsuey Cheng

Bone marrow-derived mesenchymal stem cells enhanced diabetic wound healing through recruitment of tissue regeneration in a rat model of streptozotocin-induced diabetes.

Background: This study investigated whether bone marrow–derived mesenchymal stem cell therapy has effectiveness in the enhancement of diabetic wound healing through tissue regeneration. Methods: The authors used a dorsal skin defect (6 × 5 cm) in a streptozotocin-induced diabetes rodent model. Forty male Wistar rats were divided into four groups: group I, nondiabetic rats (controls); group II, diabetic controls receiving no mesenchymal stem cells; group III, rats receiving 1 × 107 stem cells per dose (subcutaneously administered in eight areas surrounding wound margin) on day 7; and group IV, rats receiving stem cells on days 7 and 10. Wound healing was assessed clinically. Histologic examination was performed with hematoxylin and eosin staining. CD45, Ki-67, prolyl 4-hydroxylase, epidermal growth factor, and vascular endothelial growth factor were evaluated with immunohistochemical analysis. Results: Overall clinical results showed that wound size was significantly reduced in mesenchymal stem cell–treated rats as compared with controls. Complete wound-healing time was statistically shorter in rats treated once as compared with controls (6.6 ± 1.13 weeks versus 9.8 ± 0.75 weeks; p < 0.001). It was significantly shorter in rats treated with mesenchymal stem cells twice as compared with rats treated once (5.2 ± 0.75 weeks versus 6.6 ± 1.13 weeks; p = 0.026). Histologic analysis revealed significant reduction in topical proinflammatory reaction and suppression of CD45 expression in the mesenchymal stem cell group as compared with the control group. On immunohistochemistry analysis, significant increases in epidermal growth factor, vascular endothelial growth factor, prolyl 4-hydroxylase, and Ki-67 expression were noted in the treated group as compared with the control group. Conclusions: Mesenchymal stem cells significantly enhanced diabetic wound healing. Treatment with them is associated with increases of biomarkers in tissue regeneration.

Electroporation for direct spinal gene transfer in rats

We investigated the feasibility of delivering exogenous genes into spinal cord using direct in vivo electrotransfection. Gene transfer to the spinal cord was accomplished via direct intrathecal injection of pE-GFP C1 vector, followed by five electric pulses for 50 ms at 200 V delivered intrathecally. The spinal cords were retrieved and analyzed with fluorescence microscopy, reverse transcription polymerase chain reaction (RT-PCR), and Western blotting. At day 1, 3 or 7 following electroporation a clear GFP expression in spinal cord tissue was detected. The most prominent transfection occurred in the meningeal cells and superficial layer of the spinal cord. Successful transfection was also confirmed with RT-PCR and Western blotting. The expression of GFP protein was peaked between 3 and 7 days after electroporation and significantly decreased at 14 days. No behavioral or spinal neurodegenerative changes were detected at any time point. This study demonstrates that direct in vivo electrotransfection represents an effective and simple method for spinal gene delivery and have a potential to be used clinically, especially, acute or chronic pain.

Adipose-Derived Stem Cells Accelerate Diabetic Wound Healing Through the Induction of Autocrine and Paracrine Effects.

Cell-based therapy is an attractive approach for the treatment of chronic nonhealing wounds. This study investigated whether adipose-derived stem cells (ASCs) can accelerate diabetic wound healing and traffic in the engraftment of ASCs. Dorsal full-thickness skin wound defects (6 × 5 cm) were created in a streptozotocin (STZ)-induced diabetes rodent model. Group I served as a nondiabetic normal control, group II served as a diabetic control without ASCs, and group III included rats that were injected subcutaneously in the wound margin twice with nondiabetic ASCs (1 × 107 ASCs/dose). The wound healing was assessed clinically. Histological examination and immunohistochemical analyses of periwound tissue were performed. Green fluorescence protein (GFP)+-ASCs were used to examine the engraftment of these cells after injection. XenoLight DiR-labeled ASCs were implanted to detect migration ability using an IVIS imaging system. Results revealed that complete wound healing time statistically decreased in the ASC-treated group compared to the controls (p < 0.001). Histological examination revealed the ASC-treated group showed a significant reduction in the proinflammatory reaction, with significantly increased levels of EGF, VEGF, rPH, and Ki-67 expression compared to the controls. The populations of GFP+-ASCs in circulating blood significantly increased after ASC injection compared to those of controls. Immunofluorescence staining showed GFP+-ASCs significantly accumulated in the subdermal layer of the wound margin and increased angiogenesis via vWF and VEGF expression after injection. IVIS analysis revealed ASCs could exist and home into the periwound area up to 8 weeks postimplantation. In conclusion, ASCs significantly enhanced diabetic wound healing, engrafted into the local wound tissue, and implanted into circulating blood. ASC treatment stimulated neoangiogenesis and increased tissue regeneration through paracrine and autocrine mechanisms.

Intrathecal spinal progenitor cell transplantation for the treatment of neuropathic pain

Injury to, or dysfunction of, the nervous system can lead to spontaneous pain, hyperalgesia, and/or allodynia. It is believed that the number and activity of GABAergic neurons gradually decreases over the dorsal horn. Glutamic acid decarboxylase (GAD) immunocompetence has been demonstrated on spinal progenitor cells (SPCs) cultivated in vitro. The intrathecal implantation of these cultivated progenitor cells may provide a means of alleviating neuropathic pain. Chronic constriction injury (CCI) of the sciatic nerve was used to induce chronic neuropathic pain in the hind paw of rats. SPCs (1 × 106) were implanted intrathecally on the third day after the CCI surgery. The behavioral response to thermal hyperalgesia was observed and recorded during the 14 days postsurgery. Various techniques were utilized to trace the progenitor cells, confirm the differentiation, and identify the neurotransmitters involved. GAD immunoactivity was revealed for 65% of the cultivated spinal progenitor cells in our study. We also determined that transplanted cells could survive more than 3 weeks postintrathecal implantation. Significant reductions were demonstrated for responses to thermal stimuli for the CCI rats that had received intrathecal SPC transplantation. A novel intrathecal delivery with SPCs reduced CCI-induced neuropathic pain.

Preincisional subcutaneous infiltration of ketamine suppresses postoperative pain after circumcision surgery

ObjectiveN-methyl-D-aspartate and other glutamate receptors have been shown to present on the peripheral axons of primary afferents, and peripheral injection of N-methyl-D-aspartate-receptor antagonists can suppress hyperalgesia and allodynia. Thus, this study examined postoperative analgesic and adverse effects of local ketamine administered postoperatively. MethodsKetamine (0.3%, 3 mL) or saline was subcutaneously infiltrated before incision in a double-blind manner using a sample population of 40 patients undergoing circumcision surgery, equally and randomly assigned to 2 groups based on the treatment. The saline-infiltrated patients also received 9-mg intramuscular ketamine into the upper arm to control for any related systemic analgesic effects. The patients were followed up for 24 hours to determine postoperative analgesia and identify adverse effects. ResultsIn the ketamine-infiltrated patients, the time interval until first analgesic demand (166 vs. 80 min) was longer and the incidence of pain-free status (pain score=0) during movement (45% vs. 10%) and erection (40% vs. 0%) was significantly higher than for the saline-treated analogs (P<0.05). The dose of ketorolac use and pain score during erection were significant lower in group ketamine patients. No significant differences were noted with respect to the incidence of adverse effects comparing the 2 groups. DiscussionWe conclude that preincisional subcutaneous ketamine infiltration can suppress postoperative pain after the circumcision surgery.

Double-blind parallel comparison of multiple doses of apraclonidine, clonidine, and placebo administered intra-articularly to patients undergoing arthroscopic knee surgery.

Objective:This clinical study assessed and compared the potential analgesic and adverse effect of IA apraclonidine with IA clonidine. Methods:Eighty patients scheduled for arthroscopic knee surgery under general anesthesia were randomized to receive, in a double-blind manner, either IA normal saline (group 1), 50 μg IA apraclonidine (group 2), 150 μg IA apraclonidine (group 3), or 150 μg IA clonidine (group 4), all in a volume of 20 mL subsequent to surgery. Visual analog pain scores (VAS), the duration of analgesia as defined by the time to first demand for supplemental analgesics, the subsequent 24-hour consumption of postoperative supplementary analgesics, and patient adverse effects were evaluated. Results:The patients from groups 3 and 4 demonstrated a longer duration of analgesia and used fewer analgesics in the first postoperative 24 hour period compared with group 1 and 2 patients (P < 0.05). The VAS scores corresponding to the periods 1, 2, and 4 hours postoperatively were significantly lower for group 3 than for group 1 patients. The VAS scores at 1 and 4 hours postoperatively were also lower for group 3 than for group 2 patients (P < 0.05). There was no significant difference in the incidence of side effects among the 4 groups. Discussion:The IA application of 150 μg apraclonidine and 150 μg clonidine provide similar degree of postoperative analgesia following knee arthroscopic surgery without any difference in adverse events.

Immunoneutralization of c-Fos Using Intrathecal Antibody Electroporation Attenuates Chronic Constrictive Injury-induced Hyperalgesia and Regulates Preprodynorphin Expression in Rats

Background In vivo electroporation has been successfully used for the introduction of DNA, RNA, oligonucleotides, and proteins into cells for experimental and therapeutic purposes. The authors evaluated the efficacy of electroporation-mediated c-Fos antibody therapy for neuropathic pain in vitro and in vivo. Methods First, the authors studied the inhibitory effects of intrathecal c-Fos antibody electroporation on the activating protein (AP-1) promoter activity in cultured spinal neuronal cells transfected with p-AP-Luc plasmid and activated with 100 &mgr;m glutamate. The inhibitory effect of c-Fos antibody electroporation in the regulation of AP-1 promoter activity was assessed according to the relative luciferase activity. Second, rats with chronic constrictive injury underwent electroporation treatment for neuropathic pain using c-Fos antibody. Thermal nociceptive thresholds were measured before chronic constrictive injury and then on even-numbered days, up to and including day 14, to assess and compare the therapeutic effects of intrathecal electroporation. The time course was assessed by Western blot analysis and by immunohistochemical analysis. Pronociceptive gene expression was measured by assessing prodynorphin mRNA and dynorphin peptides on days 2 and 10 after intrathecal c-Fos electroporation. Results Cotransfection of c-Fos antibody significantly decreased glutamate-induced AP-1 activity. Intrathecal electrotransfer of c-Fos antibody attenuated spinal dynorphin levels, as manifested by significantly elevated pain thresholds in the chronic constrictive injury–affected limbs. Conclusion This study shows that transfer of antibody into rat spinal cords by intrathecal electroporation is a useful method to study the function of endogenous factors of spinal-related disorders.

Intrathecal Clonidine Decreases Spinal Nitric Oxide Release in a Rat Model of Complete Freund's Adjuvant Induced Inflammatory Pain

AbstractA long-lasting antihyperalgesic effect has been demonstrated for intrathecal (IT) clonidine, an alpha2-adrenergic agonist. In the present study, the mechanism and antihyperalgesic effects of IT clonidine were examined post-treatment in a rat model of Complete Freunds Adjuvant (CFA)-induced inflammatory hyperalgesia. Using a chronic model of spinal cord dialysis, we examined the effect of the adjuvant-induced inflammation on spinal release of nitric oxide (NO) and the development of chronic pain and assessed the antinociceptive effects and mechanisms of the alpha2-adrenergic agonist, clonidine (IT). Chronic, persistent inflammatory pain was induced by left hind paw injection of 0.3 ml CFA prepared in a mixture with Mycobacterium butyricum. Rats were randomly assigned to groups receiving IT clonidine in discrete doses of 1, 10 or 50 μg, 3 or 24 hr post-inflammation. Measurement of total NOx (NO +

Effect of thiopental, propofol, and etomidate on vincristine toxicity in PC12 cells

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