Dwi Liliek Kusindarta

Innervation of the rat trachea by bilateral cholinergic projections from the nucleus ambiguus and direct motor fibers from the cervical spinal cord: a retrograde and anterograde tracer study

A tract-tracer method was employed to examine the innervation of the rat trachea. Cholera toxin beta subunit (CTB) was injected into the following locations in separate groups of rats: (1) ventral trachea, (2) lateral trachea, (3) ventral trachea after the excision of the nodose ganglion, and (4) ventral trachea after the transection of C1-C2 spinal nerves. CTB injection in the ventral trachea showed bilateral labeling of neurons in the nucleus ambiguus (NA), medial subnucleus of the nucleus of the solitary nucleus, dorsal motor nucleus of the vagus (DMV), and lamina IX of C1-C6. CTB injection in the lateral trachea showed significant ipsilateral predominance of neuronal labeling in the NA and lamina IX of C1-C2 segments. CTB injection in rats after the excision of the nodose ganglion revealed no labeling in the ipsilateral DMV and NA and a significant reduction of neuronal labeling in C1. CTB injection in rats after the transection of C1-C2 spinal nerves showed a significant decrease in the number of labeled neurons in ipsilateral NA, C1, and C2 and no labeling of fibers in C1-C2. The combination of retrograde fluorogold labeling and choline acetyltransferase (ChAT) immunostaining revealed that all fluorogold-labeled neurons in the NA and lamina IX of C1-C2 colocalized with ChAT. The injection of biotinylated dextran amine in NA produced labeling in axonal terminals on postganglionic neurons, but not in other regions of the trachea. Our findings indicate that the rat trachea is innervated bilaterally by cholinergic motor neurons in NA and C1-C2, while those traveling through the spinal nerves project directly to the trachea.

Human umbilical mesenchymal stem cells conditioned medium promote primary wound healing regeneration.

Aim: This research was conducted to clarify the capability of human umbilical mesenchymal stem cells conditioned medium (HU-MSCM) to promote regenerations of primary wound healing on the incision skin injury. Materials and Methods: In this study, two approaches in vitro and in vivo already done. On in vitro analysis, tube formation was performed using HU vein endothelial cells in the presence of HU-MSCM, in some experiments cells line was incubated prior the presence of lipopolysaccharide and HU-MSCM then apoptosis assay was performed. Furthermore, in vivo experiments 12 female rats (Rattus norvegicus) were used after rats anesthetized, 7 mm wound was made by incision on the left side of the body. The wound was treated with HU-MSCM containing cream, povidone iodine was run as a control. Wound healing regenerations on the skin samples were visualized by hematoxylin-eosin staining. Results: In vitro models elucidate HU-MSCM may decreasing inflammation at the beginning of wound healing, promote cell migration and angiogenesis. In addition in vivo models show that the incision length on the skin is decreasing and more smaller, HE staining describe decreasing of inflammation phase, increasing of angiogenesis, accelerate fibroplasia, and maturation phase. Conclusions: Taken together our observation indicates that HU-MSCM could promote the acceleration of skin tissue regenerations in primary wound healing process.

Ocimum sanctum Linn. stimulate the expression of choline acetyltransferase on the human cerebral microvascular endothelial cells

Aim: This research was conducted to identify the expression of choline acetyltransferase (ChAT) in human cerebral microvascular endothelial cells (HCMECs) and to clarify the capability of Ocimum sanctum Linn. ethanolic extract to stimulate the presence of ChAT in the aging HCMECs. Materials and Methods: In this study, we perform an in vitro analysis some in the presence of an ethanolic extract of O. sanctum Linn. as a stimulator for the ChAT expression. HCMECs are divided become two groups, the first is in low passage cells as a model of young aged and the second is in a high passage as a model of aging. Furthermore to analysis the expression of ChAT without and with extract treatments, immunocytochemistry and flow cytometry analysis were performed. In addition, ChAT sandwich enzyme-linked immunosorbent assay is developed to detect the increasing activity of the ChAT under normal, and aging HCMECs on the condition treated and untreated cells. Results: In our in vitro models using HCMECs, we found that ChAT is expressed throughout intracytoplasmic areas. On the status of aging, the ethanolic extract from O. sanctum Linn. is capable to stimulate and restore the expression of ChAT. The increasing of ChAT expression is in line with the increasing activity of this enzyme on the aging treated HCMECs. Conclusions: Our observation indicates that HCMECs is one of the noncholinergic cells which is produced ChAT. The administrated of O. sanctum Linn. ethanolic extract may stimulate and restore the expression of ChAT on the deteriorating cells of HCMECs, thus its may give nerve protection and help the production of acetylcholine.

The structural and functional recovery of pancreatic β-cells in type 1 diabetes mellitus induced mesenchymal stem cell-conditioned medium

Aim: Various studies have shown that secreted factors alone in culture medium without stem cell are capable of repairing tissues by itself in various conditions involving damaged tissue/organ. Therefore, this study was aimed to investigate the role of human umbilical cord mesenchymal stem cell-derived conditioned medium (CM) on the recovery of pancreatic β-cells in Wistar rats (Rattus norvegicus) with type 1 diabetes mellitus. Materials and Methods: The 0.05 ml CM induction was applied to the diabetic group of rats in weeks 1, 2, 3, and 4. 1 week after each CM induction, insulin concentration was analyzed using ELISA. The pancreas was divided into 3 regions, processed by paraffin method, stained with hematoxylin-eosin, and immunohistochemical method for insulin. Results: This study indicated the decrease in the total number of islets and insulin concentration after the injection of single dose of alloxan. The exocrine acini were also damaged. Microscopic observation detected the presence of small islets in the diabetic group 1 week after the first 0.05 ml CM induction. The number and size of the islets increased in line with the CM doses and time of inductions. Immunohistochemically, the presence of low intensity of insulin-positive cells could be recognized at the splenic and duodenal regions of the pancreas, but not gastric region, 1 week after the first and second 0.05 ml CM induction. The intensity of staining and the number of insulin-positive cells increased dramatically in 1 week after the third and fourth 0.05 ml of CM induction in all regions of the pancreas. The data of insulin blood concentration showed clear differences between the second and the fourth induction of 0.05 ml CM induction. Conclusions: This study showed very strong evidence on the role of human umbilical cord mesenchymal stem cell-derived CM in recovering the pancreatic β-cells damage in Wistar rats (R. norvegicus) with type 1 diabetes mellitus, structurally and functionally.

The analysis of hippocampus neuronal density (CA1 and CA3) after Ocimum sanctum ethanolic extract treatment on the young adulthood and middle age rat model

Aim: This study aimed to assess the changes in neuronal density in CA1 and CA3 regions in the hippocampus of young adulthood and middle age rat model after feeding by Ocimum sanctum ethanolic extract. Materials and Methods: In this research, 30 male Wistar rats consist of young to middle-aged rats were divided into three groups (3, 6, and 9 months old) and treated with a different dosage of O. sanctum ethanolic extract (0, 50, and 100 mg/kg b.w.) for 45 days. Furthermore, cresyl violet staining was performed to analyze hippocampus formation mainly in CA1 and CA3 area. The concentrations of acetylcholine (Ach) in brain tissues were analyzed by enzyme-linked immunosorbent assay. Results: In our in vivo models using rat model, we found that the administration of O. sanctum ethanolic extract with a dosage of 100 mg/kg b.w. for 45 days induced the density of pyramidal cells significantly in CA1 and CA3 of the hippocampus. These results were supported by an increase of Ach concentrations on the brain tissue. Conclusions: The administration of O. sanctum ethanolic extract may promote the density of the pyramidal cells in the CA1 and CA3 mediated by the up-regulated concentration of Ach.

The neuroprotective effect of Ocimum sanctum Linn. ethanolic extract on human embryonic kidney-293 cells as in vitro model of neurodegenerative disease

Aim: This study aimed to analyze the neuroprotective effect of Ocimum sanctum Linn. ethanolic extract (OSE) on human embryonic kidney-293 (HEK-293) cells as the in vitro model of neurodegenerative diseases. Materials and Methods: In this research, HEK-293 cells divided into five groups consisting of normal and healthy cells (NT), cells treated with Camptothecin 500 µM as the negative control, cells treated with trimethyltin 10 µM (TMT), cells treated with OSE 75 µg/ml, and cells pre-treated with OSE 75 µg/ml then induced by TMT 10 µM (OSE+TMT). MTT assay and phase contrast microscopy were applied to observe the cell viability quantitatively and morphological after Ocimum sanctum Linn extract treatment. Finally, the reverse transcription polymerase chain reaction was employed to study the expression of choline acetyltransferase (ChAT). Results: The MTT assay and phase contrast microscopy showed that OSE pre-treatment significantly increased the viability of TMT-induced apoptotic cells and maintained cell viability of the normal HEK-293 cells. Expression of ChAT markedly reduced on TMT treatment group, but OSE administration stabilized ChAT expression in TMT-induced HEK-293 cells. Conclusion: This present study proved that OSE administration has neuroprotective effect by increased HEK-293 cells viability and maintain ChAT expression.

Ethanolic extract Ocimum sanctum. Enhances cognitive ability from young adulthood to middle aged mediated by increasing choline acetyl transferase activity in rat model

Patients with dementia are increasing steadily, cognitive impairment by dementia not only exclusively suffers by old people but also young to middle aged individuals. However, the mechanism of cognitive impairment occurs in young people is not understood. Further, current medication to impairment did not provide satisfactory results. Therefore, we investigated the potential role of Ocimum sanctum ethanolic extract to enhance cognitive ability in the rat in vivo model. Young to middle aged rats were divided into 3 groups (3, 6, 9 months old) were treated with (0, 50 and 100 mg/kg b.w.) O. sanctum for 45 days. We employed a behavioral assay to assess cognitive ability. Further, Nissl staining was performed to analyze hippocampus formation in dentate gyrus (DG), cornu ammonis 1 (CA1), cornu ammonis 3 (CA3). The expression and activity of ChAT in brain was analyzed by RT-PCR and ELISA. Our results showed that treatment of O. sanctum with a dosage of 100 mg/kg b.w. for 45 days induced the cognitive ability in nine months old rats. Further, we observed a significant increase in density of granular and pyramidal cells in DG, CA1, and CA3. These results were corroborated by an increase in the ChAT activity and gene expression in the rat model as well as HEK 293 cell culture model. Taken together, the administration of 100 mg/kg b.w. O.sanctum induced the expression of ChAT. The increased ChAT expression and activity may enhance the cognitive ability in 9 months old rats mimicking young and middle aged condition in humans.