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Objective To investigate the corneal permeability of cyclosprin A (CsA) loaded on polymeric vector after topical application.Methods The grafted copolymer chitosan-graft-cyclodextrin (CS-g-CD) was synthesized,and the physicochemical structures of the polymer were investigated using nuclear magnetic resonance spectroscopy (NMR) and fourier transform infrared spectroscopy (FT-IR).A novel CsA eye drop was prepared using the grafted copolymer as carrier material.The physicochemical properties of eye drop,including drug-loading content,osmotic pressure and viscosity were investigated by high performance liquid chromatography-mass spectrometry (HPLC-MS),osmotic pressure gauge and viscometer,respectively.New Zealand albino rabbits were randomly divided into intact cornea CsA group,epithelium debrided CsA group and epithelium debrided control group.The corneal epithelia of the left eyes was debrided in the cornea epithelium debrided group.Cornea irritation test was performed on New Zealand albino rabbits.The aqueous humor was taken and the corneas were collected at 0.5 hour and 1 hour after instilled.The concentration of CsA was measured by HPLC-MS.Cy5 labeled vector loaded with Coumarin 6 served as model copolymers system,the penetration capabilities of the double fluorescent labeling copolymers system were monitored in vivo using two-photon scanning fluorescence microscopy on murine corneas after topical application.The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results The polymer of CS-g-CD was successfully synthesized and confirmed using NMR and FT-IR.The drug loading of CsA in eye drop solution was 0.06 ％;the osmotic pressure was 305 mOsmol/kg and the viscosity was 36.5 cP.The CsA drug delivery system had a reversible temperature-sensitive drug release behavior and had no obvious irritation on the eyes of New Zealand rabbits.One hour after treatment,the concentration of CsA in the cornea and aqueous humor of epithelium debrided CsA group was (5.88 ± 1.46) μg/g and (149.19 ± 3.93) ng/ml,respectively,which was significantly higher than (3.98 ±0.95) μg/g and (30.25± 11.43) ng/ml in epithelium debrided control group (both at P＜0.05);the concentration of CsA in the aqueous humor of intact cornea CsA group was (7.23 ± 1.31)ng/ml,which was significantly lower than that in epithelium debrided CsA group (P＜0.05).Polymer vectors were mainly retained in the corneal epithelium,and coumarin 6 gradually diffused into the deep corneal stroma with time.Conclusions The grafted copolymer can load CsA,and the eye drop can effectively overcome the corneal barrier and increase the corneal permeability of CsA.
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Objective To investigate the corneal permeability of cyclosprin A (CsA) loaded on polymeric vector after topical application. Methods The grafted copolymer chitosan.graft.cyclodextrin ( CS.g.CD ) was synthesized, and the physicochemical structures of the polymer were investigated using nuclear magnetic resonance spectroscopy ( NMR) and fourier transform infrared spectroscopy ( FT.IR) . A novel CsA eye drop was prepared using the grafted copolymer as carrier material. The physicochemical properties of eye drop,including drug.loading content, osmotic pressure and viscosity were investigated by high performance liquid chromatography.mass spectrometry ( HPLC.MS) ,osmotic pressure gauge and viscometer,respectively. New Zealand albino rabbits were randomly divided into intact cornea CsA group, epithelium debrided CsA group and epithelium debrided control group. The corneal epithelia of the left eyes was debrided in the cornea epithelium debrided group. Cornea irritation test was performed on New Zealand albino rabbits. The aqueous humor was taken and the corneas were collected at 0. 5 hour and 1 hour after instilled. The concentration of CsA was measured by HPLC.MS. Cy5 labeled vector loaded with Coumarin 6 served as model copolymers system, the penetration capabilities of the double fluorescent labeling copolymers system were monitored in vivo using two.photon scanning fluorescence microscopy on murine corneas after topical application. The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission. Results The polymer of CS.g.CD was successfully synthesized and confirmed using NMR and FT.IR. The drug loading of CsA in eye drop solution was 0. 06 ％;the osmotic pressure was 305 mOsmol/kg and the viscosity was 36. 5 cP. The CsA drug delivery system had a reversible temperature.sensitive drug release behavior and had no obvious irritation on the eyes of New Zealand rabbits. One hour after treatment,the concentration of CsA in the cornea and aqueous humor of epithelium debrided CsA group was (5. 88±1. 46)μg/g and (149. 19±3. 93)ng/ml,respectively,which was significantly higher than (3. 98±0. 95)μg/g and (30. 25±11. 43)ng/ml in epithelium debrided control group (both at P<0. 05);the concentration of CsA in the aqueous humor of intact cornea CsA group was ( 7. 23 ± 1. 31 ) ng/ml, which was significantly lower than that in epithelium debrided CsA group ( P<0. 05 ) . Polymer vectors were mainly retained in the corneal epithelium, and coumarin 6 gradually diffused into the deep corneal stroma with time. Conclusions The grafted copolymer can load CsA,and the eye drop can effectively overcome the corneal barrier and increase the corneal permeability of CsA.
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10.3969/j.issn.2095-4344.2013.25.002
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Objective To investigate the expression and the clinical significance of Lewis y antigen and Mucin 1 (MUC1),as well as to evaluate the correlation between them in epithelial ovarian tumor.Methods The expression of Lewis y antigen and MUC1 in 60 cases of epithelial ovarian malignant tumors,30 cases of borderline ovarian tumors,30 cases of benign ovarian tumors and 20 cases of normal ovarian tissues were detected by immunohistochemical staining.The relationship between Lewis y antigen and MUC1,and their relationship with biology characteristic of ovarian carcinoma were analyzed.An immunofluorescence double labeling methods was performed to detect the correlation between Lewis y antigen and MUC1.Results In malignant epithelial ovarian tumors,the positive rates of Lewis y antigen was 88.33％,which was significantly higher than the positive rates in borderline(60.00％,x2 =9.6405,P ＜0.01) and benign ovarian tumors(33.33％,x2 =28.8095,P ＜0.01) and normal ovarian samples (0,x2 =52.3457,P ＜ 0.01).The positive rates of Lewis y antigen had nothing to do with the clinical pathological parameters of ovarian tumor,but the expression intensity of Lewis yantigen was increased with the development of the malignant degree(P ＜ 0.05).The positive rates of MUC1 in malignant epithelial ovarian tumors was also significantly higher than that in borderline,benign ovarian tumors and normal ovarian samples (86.67％ vs 53.33％,30.00％,25.00％,x2 =12.0321,29.4064,27.8464 ; P ＜0.01).And the expression intensity of MUC1 also increased with the development of clinical stage(P ＜0.01),but had nothing to do with the lymph node metastasis and histological grade(P ＞ 0.05).In ovarian cancer,both Lewis y antigen and MUC1 were highly expressed,and their expression levels were positively correlated (r =0.707,P ＜0.01),and Lewis y antigen colocalized with MUC1.Conclusion Both Lewis y antigen and MUC1 are associated with the occurrence and development of ovarian cancer.Lewis y antigen and MUC1 might be a sigh of biological behavior in ovarian cancers,and this study provides theoretical evidence of ovarian cancer biological treatment.
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Objective To explore the significance of palpation-negative breast tumor resection with ultrasound-guided methylene blue labeling and percutaneous suture traction.Methods 46 cases of small breast tumors with the diameter from 0.5 cm to 1.0 cm were double labeled with methylene blue under the guide of ultrasound l hour preoperatively.The tumors were fixed,drew outward with percutaneous suture and resected accurately.All the 46 cases were rechecked by ultrasound to verify whether residual or false resection occurred 1 month later.Results All the 58 tumors of the 46 patients were accurately resected.No residue or false resection occurred.The average operation duration was 10 min to 20 min.Conclusion Palpable-negative breast tumor resection with ultrasound-guided methylene blue labeling and percutaneous suture traction has the advantages of small invasion,accurate positioning and short operation duration.
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The spicoreticulocerebellar (SRC) tract is an indirect spinocerebellar tract formed by the reticular formation (RF), which is connected to the cerebellum and spinal cord. The RF receives ascending fibers to both the spinal enlargement and sends descending fibers to the cerebellum. This study demonstrated that the connectivity of the neurons in the RF is concerned to the cerebellum and spinal cord using the anterograde projection with biotinylated dextran amine (BDA) and retrograde labeling with wheat germ agglutinin-horseradish peroxidase (WGA-HRP). Until now, a preliminary study in mammals has dealt with the afferent and efferent pathways in separating groups of neurons in the RF. There are only few reports on chickens. This study examined the SRC tract in chickens. Following bilateral injections we injected BDA into chicken spinal cord (lumbosacral enlargement) and WGA-HRP into the cerebellum. Both of single- and double-labeled cells were found within the RF. The spinoreticular axons were mainly distributed from the potomedullary junction to the rostral medulla in the rostro-caudally RF levels, for example, nucleus of reticularis (n. r.) pontis oralis, locus coeruleus, n. r. pontis caudalis, n. r. pars gigantocellularis, n. r. gigantocellularis and n. r. parvocellualris. Reticulocerebellar labeling by the WGA- HRP was found in the same place as well as that of the BDA-projection. We observed that the proportion and location of double labeling cells in the chicken were almost similar in each level, comparing to the rodents. These results suggest that the reticular formation is strongly related to the spicoreticulocerebellar tract in chickens.
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Animals , Afferent Pathways/physiology , Biotin , Cerebellum , Chickens , Dextrans , Efferent Pathways/physiology , Microinjections , Reticular Formation , Spinal Cord , Wheat Germ Agglutinin-Horseradish Peroxidase ConjugateABSTRACT
Apodemus agrarius has been using for experimental purpose to identifying the route of infection and pathogenesis of korean hemorrhagic fever. However, despite the increasing amount of information being published at present about the physiologic and ecologic characteristics of Apodemus, few data are availalle about the morphologic findings in the brain. The NADPH-diaphorase[NADPH-d] positive neurons, uniquely resistant to toxic insults and neurodegenerative diseases, have been colocalized with neurons in the brain and peripheral tissue containing nitric oxide synthase, which generates nitric oxide, a recently identified neuronal messenger molecule. In this study we used NADPH-d histochemistry to evaluate the distribution of neuropeptide Y-immunoreactive[NPY-IR] cells within neurons which contain nitric oxide synthase. In the cerebral cortex of Apodemus agrarius, NADPH-d positive and NPY-IR neurons were observed in all cortical layers, but they were concentrated in two bands layer II/III and V/VI extending into the subcortical white matter. Double labeling for NADPH-d and NPY showed colocalization of NPY with NADPH-d in numerous neurons of the cerebral cortex. The data obtained showed that about 74-79% of NPY-IR neurons contained NADPH-d in the neocortex and that 77-89% in the allocortex. The number of NPY-IR/NADPH-d positive neurons was about 10-13 per unit area [2,500,000 micrometer] in the neocortex and about 11-25 in the allocortex except retrosplenial cortex. In the retrosplenial cortex, the number of double labeled neurons was about 5 per unit area. NPY-IR and NADPH-d positive neurons were predominantly medium-sized with extended, multipolar or bipolar dendritic branches which belong to fusiform or stellate cell types. A moderately dense network of fine, varicose NADPH-d positive fibers was present throughout all cortical layers.
Subject(s)
Animals , Brain , Cerebral Cortex , Hemorrhagic Fever with Renal Syndrome , Murinae , Neocortex , Neurodegenerative Diseases , Neurons , Neuropeptide Y , Neuropeptides , Nitric Oxide , Nitric Oxide SynthaseABSTRACT
The relationship between CCK-and VP-neurons in the rat retrochiasmatic area was studied at ultrastructural level by means of pre-embedding(PAP) double immunoelectron microscopic labeling technique. First, the VP-immunoreactivity was demonstrated by DAB method. After thoroughly washing, the CCK-immuno- reactivity was revealed by ammonium molybdate-TMB method. Being stabili- zed by DAB-cobalt chloride, the sections were embedded in Epon 812. Under ele- ctron microscope, it was observed that in the retrochiasmatic area, the VP-LI products distributed diffusely as high electron dense granular or flocculent depo- sits, whereas the CCK-LI products distributed sparsely as needle-or mass-like deposits. VP-LI perikarya were small in size with oval shape and CCK-LI peri- karya were medium in size with polygonal shape. CCK-LI perikarya and dendri- tes received afferent synapses from non-CCK- and non-VP-axonal terminals VP- LI axons received afferent synapses from VP and non-VP-axonal terminals It was interesting that the VP-LI axonal terminals formed efferent axoaxonic syna- pses with CCK-LI axonal endings and, vice versa, the CCK-LI axonal terminals established also efferent axoaxonic synapses with VP-LI axonal endings. The above mentioned results identified for the first time that in the rat retrochiasma- tic area not only there were CCK- and VP-neurons, but also there were reciprocal synaptic regulations between above two kinds of peptidergic neuron, providing new ultrastructural basis for the regulatory mechanism of the neuroendocrine in hypothalamus.
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Objective To investigate the distribution of interleukin\|2 receptor (IL\|2R) and glucocorticoid receptor(GR) and identify coexistence of IL\|2R and GR in the rat brain. Methods The double labeling immunocytochemical technique(PAP method combined with ABC method), DAB and BDHC were used in the double labeling immunocytochemical method as the chromogens respectively. The reactive products of former was brown or yellow and later was black blue. Results IL\|2R and GR positive neurons were widely distributed in the cerebral cortex, hippocampus, thalamus, many motor and sense nuclei in the brain stem. The immunoreactive products of IL\|2R were found to be located on cell membrane and GR in nucleus and cytoplasm. There were a lot of positive double labeling neurons in the rat brain. The rate of double labeled cells in the total number of positive cells varied in different regions of brain, such as, 50 percent in cerebral cortex and 30 percent in nucleus of abducent nerve. Conclusion Immunogical cytokines and hormone could regulate the neuronal function through their corresponding receptors which coexisted in the same brain neurons. The present study might provide morphological evidence in the level of receptor for the immuno\|neuro\|endocrine network.
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The distribution of neurotensin (NT) and neuropeptide Y (NPY) in the rat hypothalamic arcuate nucleus has been studied ultrastructurally by means of double labeling preembedding immunoelectron microscopic PAP technique. First, the NPY immunoreaction was demonstrated by chromogen DAB, and second, the NT immunoreaction was demonstrated by ammonium molybdate-TMB method. After being stabilized by DAB-cobalt chloride, the vibratome sections were processed for electron microscopic study. The results showed that in the arcuate nucleus the NPY immunoreactive products appeared as high electron-dense granular or flocculent materials deposited diffusely in the organelles and matrix of perikaryon, around the dendritic microtubules and axonic small clear vesicles. Whereas the NT immunoreactive products were dense needle- or mass-like deposits distributed dispersively in the perikaryon, dendrites and axon terminals. They can easily be distinguished although being intermingled together. The NPY-containing dendrites and axons formed synaptic connections with immuno-negative axon terminals, NT-containing somata and dendrites formed also synaptic conections with negative axon terminals. In addition, NPY-positive axon terminals formed symmetrical axodendritic synapses with NT-positive dendrites. The present results provided another new ultrastructural evidence for the peptidergic synaptic regulation of NT neurons in hypothalamus.
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Divergent axon collaterals from forebrain and brainstem to the dorsal hippocampus in the rat were examined by means of HRP method and fluorescent retrograde double labeling method.HRP solution was injected into the left or right dorsal htppocampus of 3 rats. HRP labeled cells were observed in the bilateral medial parts of medial septal areas, vertical limbs of the diagonal band of Broca, supramammillary nuclei, submammillothalamic nuclei, superior central nucleus and locus ceruleus.PI and Bb solution were injected into the left and right hippocampus of 3 rats separately. Many retrograde double labeled cells were found in the nuclei mentioned above.The results indicate that the retrograde double labeled ceils in these nuclei may divergently projected to both the left and right dorsal hippocampi. They play an important role in harmonizing the activity of bilateral hippocampi.
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Objective To investigate the synaptic connections between endomorphin-immunoreactive(ir) terminals and ? opioid receptor-ir neurons in the superficial layers(laminae Ⅰ and Ⅱ) of the rat spinal dorsal horn. Methods The double-labeling immunohistochemical electron microscopic technique,i.e.pre-embedding immunohistochemical detection of endomorphin combin with pre-embedding immuno-gold particles labeling technique to identify ? opioid receptor-immunoreactivity,was used in the present study. Results ? opioid receptor-ir neuronal cell bodies,fibers as well as axon terminals were observed in laminae Ⅰ and Ⅱ of the spinal dorsal horn,meanwhile dense endomorphin-ir axon terminals were located mainly within the same region.Under electron microscope,the synaptic connections formed by endomorphin-ir axon terminals labeled with diaminobenzidine(DAB) reaction products and ? opioid receptor-ir neuronal cell bodies and dendritic profiles labeled with immuno-gold particles were observed.The principle synaptic type was asymmetric synapse.There were more axon-dendritic synapses than axon-somatic ones as encountered in the present study.Conclusion The distribution patterns of endomorphin-ir and ? opioid receptor-ir structures match with each other in the superficial laminae of the spinal dorsal horn,that provides morphological base for the antinociceptive effects of endomorphin and ? opioid receptor in the processing of nociceptive information transmission.