In situ epicatechin-loaded hydrogel implants for local drug delivery to spinal column for effective management of post-traumatic spinal injuries

Purpose: To prepare hydrogels loaded with epicatechin, a strong antioxidant, anti-inflammatory, and neuroprotective tea flavonoid, and characterise them in situ as a vehicle for prolonged and safer drug delivery in patients with post-traumatic spinal cord injury. Methods: Five in situ gel formulations were prepared using chitosan and evaluated in terms of their visual appearance, clarity, pH, viscosity, and in vitro drug release. In vivo anti-inflammatory activity was determined and compared with 2 % piroxicam gel as standard. Motor function activity in a rat model of spinal injury was examined comparatively with i.v. methylprednisolone as standard. Results: The N-methyl pyrrolidone solution (containing 1 % w/w epicatechin with 2 to 10 % w/w chitosan) of the in situ gel formulation had a uniform pH in the range of 4.01 ± 0.12 to 4.27 ± 0.02. High and uniform drug loading, ranging from 94.48 ± 1.28 to 98.08 ± 1.24 %, and good in vitro drug release (79.48 ± 2.84 to 96.48 ± 1.02 % after 7 days) were achieved. The in situ gel prepared from 1 % epicatechin and 2 % chitosan (E5) showed the greatest in vivo anti-inflammatory activity (60.58 % inhibition of paw oedema in standard carrageenan-induced hind rat paw oedema model, compared with 48.08 % for the standard). The gels showed significant therapeutic effectiveness against post-traumainduced spinal injury in rats. E5 elicited maximum motor activity (horizontal bar test) in the spinal injury rat model; the rats that received E5 treatment produced an activity score of 3.62 ± 0.02 at the end of 7 days, compared with 5.0 ± 0.20 following treatment with the standard. Conclusion: In situ epicatechin-loaded gel exhibits significant neuroprotective and anti-inflammatory effects, and therefore can potentially be used for prolonged and safe drug delivery in patients with traumatic spinal cord injury.

Behavioural studies on the ethanol leaf extract of Grewia carpinifolia in Wistar rats.

Background: Grewia carpinifolia is a plant commonly used in the tropics to manage various central nervous system (CNS) disorders. However, despite its widespread use no scientific work has been reported to validate these claims. Objectives: To evaluate the activity of G. carpinifolia as it affects behaviour using animal model. Methods: Twenty five adult Wistar rats were randomly divided into five groups (A-E). Group A served as control (given only distilled water), Groups B,C, D and E were administered with single oral dose of ethanol extract of G. carpinifolia leaf at 100, 200, 400 and 800 mg/kg body weight respectively for twenty eight days consecutively. Subsequently, open field test, negative geotaxis and hanging wire test were performed. Body and brain weights were measured and histological examination of the brain was also performed. Results: At the tested doses, the extract significantly increased the time spent on the hanging wire and decreased locomotor activity at 800 mg/kg. No significant difference was observed in body and brain weights of extract treated groups when compared with the control. No visible histological lesion was also observed. Conclusion: The plant extract may improve muscular strength at tested doses and possess CNS depressant activity at 800 mg/ kg.

Early Adaptation of Small Intestine After Massive Small Bowel Resection in Rats

Background: It is important that the residual bowel adapts after massive resection. The necessary intestinal adaptation is a progressive recovery from intestinal failure through increase in absorptive surface area and functional capacity and includes both morphological and functional adaptations. Objectives: The aim of this study was to investigate intestinal morphological and functional adaptations of small bowel syndrome (SBS) model rats (SBS1W) 7 days after bowel resection. Materials and Methods: Male sprague–dawley rats (n = 20/group) underwent either a 75% proximal small bowel resection (SBS1W group) or a control operation (control group). Markers of morphological adaptation were revealed by TEM analysis of H&E-stained tissue samples. The intestinal barrier condition was assessed by BT, and sIgA concentration in intestinal mucus was measured by ELISA. Contractility and the slow wave rhythm of the entire intestinal remnant were measured and recorded. Results: The SBS1W group experienced more weight loss than control group and had a clearly different intestinal morphology as revealed in TEM images. Compared with control rats, the SBS1W group had a lower sIgA concentration in intestinal mucus and higher BT to lymph nodes (70% vs 40%; level I), portal blood (40% vs 10%; level II), and peripheral blood (60% vs 30%; level III). Disorder of spontaneous rhythmic contraction, irregular amplitude, and slow frequency were detected in the SBS1W group by a muscle strips test. Similarly, the slow wave of the entire intestinal remnant in the SBS1W group was irregular and uncoordinated. Conclusions: The finding of intestinal adaptation following massive SBR in SBS1W rats provides more understanding of the mechanisms of progressive recovery from the intestinal failure that underlies SBS. The mechanical, chemical, immunological, and biological barriers were all impaired at 7 days following bowel resection, indicating that the SBS model rats were still in the intestinal adaptation phase.

Effect of recombinant human erythropoietin expressions of apoptosis genes in rats following traumatic brain injury

Purpose: To explore the effect of recombinant human erythropoietin (r-HuEPO) on apoptosis in rats after traumatic brain injury. Methods: A total of 48 traumatic brain-injured Sprague Dawley (SD) rats were obtained by improved Feeney’s traumatic brain injury model, and were randomly divided into four groups: normal salinetreated rats (control) and rats treated with r-HuEPO at doses of 1000 U/kg, 3000 U/kg and 5000 U/kg. Brain tissues were collected on the 7th day after trauma surgery. Apoptotic cells, and NF-kappa B (NFĸB)-, c-myc-, and Fas/Fasl-positive cells were identified in brain tissues by immunohistochemical assay. Results: After treatment with r-HuEPO (3000 and 5000 U/kg), expression of NF-κB and Fas/Fasl were significantly decreased (p < 0.05) compared to control rats, especially at the 5000 U/kg dose (p < 0.01). However, for c-myc, no significant difference was observed between r-HuEPO treatment and control groups (p > 0.05). Compared to the 1000 U/kg r-HuEPO group, Fas/Fasl expression levels were significantly lower in the 3000 and 5000 U/kg r-HuEPO groups (p < 0.05). Additionally, expression of NF-κB and Fasl in the 5000 U/kg r-HuEPO group was significantly lower than that in the 3000 U/kg r- HuEPO group (p < 0.05). Moreover, the number of apoptotic cells in the r-HuEPO group (5000 U/kg) was significantly lower than in the control group (p < 0.05). Conclusion: Thus, r-HuEPO may be beneficial for treating traumatic brain injury via inhibition of NFkappa B and Fas/Fasl expressions.

Healing effect of Terminalia chebula Retz extract on second-degree burns in rats

Purpose: To investigate the healing effect of Terminalia chebula Retz Extract (TCRE) on seconddegree burns in rats. Methods: Male Sprague Dawley (SD) rats, weighing 200 – 220 g, were subjected to deep seconddegree skin burns by electrical scald instrument. The animals were divided into three groups as follows: (1) second-degree burns model (control) group, (2) burns model treated with 1 % silver sulfadiazine (SSD) group, and (3) burns model treated with 100 mg·mL-1 TCRE group. On days 3, 7 and 14 following the administration of the drug/extract, the wound area and histopathological changes in rat epidermis were evaluated for the various groups. The minimum inhibitory concentration (MIC) of TCRE on Staphyloccocus aureus, Pseudomonas aeruginosa and Escherichia coli were also assessed separately. Results: On day 14, the mean wound area of TCRE treatment group (0.25 ± 0.06 cm2) was significantly smaller than that of the control rats (2.71 ± 0.20 cm2, p < 0.01). The histological results indicate that the inflammatory cells disappeared and were replaced by new granulation tissue in the group treated with 100 mg·mL-1 TCRE by day 14. Compared with SSD group rats, the inflammatory cells and fibroblast and granulation tissues of burnt rats treated with 100 mg·mL-1 TCRE were same as those of rats that had no burns. The antibacterial results revealed that the MIC of TCRE on Staphyloccocus aureus, Pseudomonas aeruginosa and Escherichia coli was 3.13, 12.5 and 6.25 mg·mL-1, respectively. Conclusion: Terminalia chebula Retz. has potentials to be developed as an effective medicinal herb for the treatment of second-degree burns.

Effect of Lycii fructus polysaccharides on ovulation failure in rats

Purpose: To investigate the effect of Lycii fructus polysaccharides (LFPS) on ovulation failure. Methods: A rat model of ovulation failure was established by intragastric administration of hydroxyurea (300 mg/kg). Rats with ovulation failure then received LFPS via oral administration at doses of 100, 200, or 400 mg/kg. The body, uterus and ovary of each rat were weighed using electronic scales. The hypothalamic-pituitary-ovarian (HPO) axis hormones, including estradiol (E2) level, follicle-stimulating hormone (FSH) activity, and luteinizing hormone (LH) activity in the serum of each rat were determined by enzyme-linked immunosorbent assay (ELISA). The levels of pro-apoptotic proteins (Fas, FasL, FADD, c-caspase-8, c-caspase-10, c-caspase-3, c-caspase-6, and c-caspase-7) in the ovarian tissue of each rat were detected by western blot. Results: Hydroxyurea reduced significantly (p < 0.01) uterus and ovary indices (uterus or ovary weight/body weight) (0.119 and 0.026 %), E2 level (3.42 pmol/L), and FSH and LH activities (2.28 and 2.76 U/L), compared with those in the normal group (0.169 and 0.039 %; 6.72 pmol/L; 2.76 and 3.75 U/L). Hydroxyurea increased significantly (p < 0.01) the levels of the above-mentioned pro-apoptotic proteins relative to those in the normal group. LFPS (100, 200, or 400 mg/kg) reversed significantly (p < 0.05 or 0.01) the effect of hydroxyurea on all of the above indices. Conclusion: LFPS exhibits a protective effect on hydroxyurea-induced ovulation failure by regulating the HPO axis hormones and death receptor-mediated apoptotic pathway.

Cardioprotective effects of Dan-Yang-Fu-Xin decoction on chronic heart failure in rats

Purpose: To evaluate the cardioprotective effects and possible mechanisms of Dan-Yang-Fu-Xin decoction (DYFX) in a rat chronic heart failure (CHF). Methods: A CHF rat model induced by ligation of the left anterior descending coronary artery was used to investigate the cardioprotective effects of DYFX. After intragastric administration for 8 weeks, several functional cardiac indices, including fractional shortening (FS), ejection fraction (EF), heart rate (HR) and cardiac output (CO) were assessed by ultrasound examination. Subsequently, inflammatory markers, viz, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), myocardial enzymes, namely, lactate dehydrogenase (LDH) and creatine kinase (CK), were also assessed by enzyme-linked immunosorbent assay (ELISA). Results: Intragastric administration of DYFX (200, 400 and 600 mg/kg) significantly reversed the decrease in body weight and increase in cardiac weight (p < 0.05) induced by CHF. Treatment with DYFX also significantly reversed EF, FS, HR, and CO changes in CHF rats. In addition, DYFX inhibited the two inflammatory cytokines (TNF-α and IL-6) and myocardial enzymes (CK and LDH), suggesting that these effects may include the mechanisms of cardioprotectiion involved in attenuation of CHF. Conclusion: DYFX possesses cardioprotective effects involving CHF. The protective mechanisms may include the suppression of expression of inflammatory mediators and myocardial enzymes.

Analgesic synergism of gabapentin and carbamazepine in rat model of diabetic neuropathic pain

Purpose: To evaluate synergy in the analgesic effects of a combination therapy of carbamazepine (CBZ) and gabapentin (GBP) in diabetic neuropathic pain. Methods: Neuropathic pain was produced in rats by a single intraperitoneal injection of streptozotocin (STZ) at 60 mg/kg. CBZ, GBP, and their combination were orally administered at varying doses (GBP 30 - 180 mg/kg; CBZ 20 - 40 mg/kg) comparable to their therapeutic doses in humans. Nociceptive responses in the diabetic rats were assessed using hot plate test. Results: Hot plate latency significantly increased with oral administration of GBP at a dose of 180 mg/kg when compared with control group (p < 0.05), while at a dose of 90 mg/kg, the increase was not significant. Oral administration of CBZ at doses of 20 and 40 mg/kg did not produce any significant impact on hot plate latency. However, a combination of GBP at 90 mg/kg and CBZ at 20 mg/kg produced significant increase in latency, compared with control group and other groups (p < 0.05), except the group that received 180 mg/kg GBP. The combination of low dose GBP 30 mg/kg and carbamazepine 30 mg/kg had no significant effect on latency (p > 0.05). Conclusion: The results obtained in this study provide useful information on the combination therapy of GBP and CBZ, which may be applied in the treatment of pain in diabetic neuropathy.

Protective effect of Rhizoma drynariae extract on osteoporosis in ovariectomized rat model

Purpose: To investigate the therapeutic effect of Rhizoma drynariae extract (RDE) on ovariectomyinduced osteoporosis in rats. Methods: Female Sprague-Dawley rats were randomly assigned to a sham-operated group (control) and five ovariectomy (OVX) subgroups: OVX with vehicle (OVX), OVX with 17ß-estradiol (E2, 25 μg/kg/day), and OVX with RDE doses (40, 80, and 160 mg/kg/day). Daily oral administration of E2 or RDE started 4 weeks after OVX and lasted for 16 weeks. The bone mineral density (BMD) of the L4 vertebrae and right femurs was estimated. The length of each femur was measured with a micrometer gauge, and the center of the diaphysis determined. Three representatives L4 vertebrae were selected to evaluate the trabecular microarchitecture. Serum alkaline phosphatase (ALP), urinary calcium (U-Ca), urinary phosphorus (U-P), urinary creatinine (Cr) and osteocalcin (OC) levels were measured. Results: The study showed that high-dose of RDE significantly inhibited the bone mineral density (BMD) reduction of L4 vertebrae (0.20 ± 0.02 g/cm3, p < 0.05) and femurs (0.18 ± 0.02 g/cm3, p < 0.05) caused by OVX and prevented the deterioration of trabecular microarchitecture (p < 0.05), which were accompanied by a significant decrease in skeletal remodeling (p < 0.05) as evidenced by the lower levels of bone turnover markers. High-dose of RDE improved morphometric parameters, namely, Tb-N (3.8 ± 0.2 mm, p < 0.05), Tb-Th (0.083 ± 0.011 mm, p < 0.05) and Tb-Sp (0.19 ± 0.01 mm, p < 0.05) in L4 vertebrae significantly. The present study indicates that the administration of RDE at higher doses over a 16-week period can prevent OVX-induced osteoporosis in rats without hyperplastic effects on the uterus. Conclusion: Thus, RDE is a potential natural alternative for postmenopausal osteoporosis treatment in elderly women.

Effect of Astragalus membranaceus (Fisch) Bunge extract on streptozocin-induced diabetic in rats

Purpose: To investigate the effect of Astragalus membranaceus (Fisch.) Bunge. extract (AMBE) on streptozotocin-induced diabetic rats. Methods: The aqueous extract of AMB was obtained by steeping the dried Astragalus membranaceus (Fisch.) Bunge. in water at 60 oC three times, each for 1 h, before first drying in an oven at 100 oC and then freeze-drying the last extract thus obtained. Diabete model rats was induced by a single intraperitoneal injection of a freshly prepared solution of streptozotocin (50 mg/kg). The rats were randomly divided into 6 groups of ten rats each: negative control group, normal control group, reference group (glibenclamide1 mg/kgbody weight) as well as AMB extract groups, namely, 40, 80 and 160 mg/kg body weight. Antihyperglycemic effect was measured by blood glucose and plasma insulin levels. Oxidative stress was evaluated in liver and kidney by antioxidant markers, viz, lipidperoxidation (LPO), superoxide dismutase (SOD), reduced glutathione (GSH), glutathione peroxidase (GPx) and catalase (CAT), while blood serum levels of creatinine and urea were also determined in both diabetic control and treated rats. Results: Compared with diabetic rats, oral administration of AMBE at a concentration of 160 mg/kg daily for 30 days showed a significant decrease in fasting blood glucose (109.438 ± 3.52, p < 0.05) and increased insulin level (13.96 ± 0.74, p < 0.05). Furthermore, it significantly reduced biochemical parameters (serum creatinine, 0.86 ± 0.29, p < 0.05) and serum urea (45.14 ± 1.79, p < 0.05). The treatment also resulted in significant increase in GSH (49.21 ± 2.59, p < 0.05), GPx (11.96 ± 1.16, p < 0.05), SOD (14.13 ± 0.49, p < 0.05), CAT (83.25 ± 3.14, p < 0.05) level in the liver and kidney of diabetic rats. Conclusion: The results suggest that AMBE may effectively normalize impaired antioxidant status in streptozotocin-induced diabetes in a dose-dependent manner. AMBE has a protective effect against lipid peroxidation by scavenging free radicals and is thus capable of reducing the risk of diabetic complications.