Application of gene chip joint pyrosequencing technology in the newborn genetic deafness gene mutation screening / 中国耳鼻咽喉头颈外科

OBJECTIVE To study the gene chip joint pyrosequencing technology in the newborn genetic deafness gene mutation screening, and provide a theoretical basis for the early diagnosis and prevention of genetic deafness. METHODS 2000 Neonatal EDTA umbilical cord blood was collected and genomic DNA (gDNA) was extracted. Microarray chip was used to detect four deafness gene at 9 mutation sites. And the positive result of gene chip detection was verified by pyrosequencing.RESULTS Among the GJB2 mutations, there were 1 case of 35delG mutation type, 3 cases of 176 del16 mutation type, 57 cases of 235del C mutation type, 9 cases of 299 del AT mutation type, 6 cases of GJB3 gene 538C>T mutation type. There were 5 cases of 1555A>G mutations and 1 case of 1494C>T mutations in mitochondrial 12S rRNA. There were 6 cases of 2168A>G mutation type and 23 cases of IVS7-2A>G mutations in SLC26A4. 103 cases of newborns carry the mutated gene in 2,000, the gene mutation rate is 5.15%. CONCLUSION All the four genes mutation at nine mutation sites are found in newborns with family history of non-hereditary deafness, and GJB2 gene mutation is common. The screening of genetic deafness in newborns is very important for early diagnosis and prevention of hereditary hearing loss. In particular, the diagnosis of mitochondrial 12S rRNA gene mutation can prevent the occurrence of deafness caused by drug use, for the genetic mutation of these carriers' health is of great significance.

Changes of the nitric oxide synthase-positive and nestin-positive neurons in the basal forebrain of castrated adult male rats following androgen replacement therapy / 中国组织工程研究

BACKGROUND: The neurons in the medial septum (MS), vertical and horizontal limbs of diagonal band of Broca (vDB and hDB) in the basal forebrain contain rich androgen receptors (ARs) and estrogen receptors (ERs) by which androgen and estrogen can act dramatically on the neurons in the basal forebrain, subsequently affecting learning and memory processes.OBJECTIVE: To qualitatively and quantitatively investigate the effects of androgen replacement therapy on the nitric oxide synthase (NOS)-positive and nestin-positive neurons in the MS, vDB and hDB of castrated adult male rats.DESIGN: A randomly controlled study on experimental animals.SETTING: Department of Anatomy and Brain Research Laboratory of Zhngshan Medical College of Sun Yat-sen University.MATERIALS: The experiment was performed at Department of Anatomy and Brain Research Laboratory of Zhongshan Medical College of Sun Yatsen University from June 2001 to June 2002. Totally twenty-eight adult male Sprague-Dawley rats were randomly divided into four groups with seven rats in each group: androgen replacement therapy for 4 weeks following 24 hours of castration (ART1), androgen replacement therapy for 2 weeks following 2 weeks of castration (ART2), vehicle replacement therapy for 4weeks following 24 hours of castration (VRT), sham-operated group (Sham).INTERVENTIONS: ① ART1 group: The castrated rats received subcutaneous injection of testosterone proprionate (25 mg/kg) dissolved in 100 μL of sterile sesame oil every other day from 10:30 am to 11:00 am for 14 times (4 weeks). ② ART2 group: The castrated rats received subcutaneous injection of testosterone proprionate with the same dosage and method as ART1 group for 7 times (2 weeks). ③ The rats in VRT group received subcutaneous injection of 100μL of sterile sesame oil for 14 times (4 weeks) by the same regime as described above. ④ Rats in Sham group only received sham-operated treatments, and testes were intact and lived for 4 weeks.MAIN OUTCOME MEASURES: Morphology and counts of NOS-positive and nestin-positive neurons were observed in the MS, vDB and hDB with immunohistochemical method at various time points.RESULTS: Data of totally 28 rats were involved in the final analyses. ①Morphological features of both NOS-positive and nestin-positive neurons in the MS, vDB and hDB were not significantly changed among four groups. ② The number of NOS-positive and nestin-positive neurons in the MS and vDB of VRT group were significantly higher than those of Sham group (P＜ 0.05 or 0.01), whereas the numbers of the NOS-positive and nestin-posi-tive neurons in the MS and vDB of ART1 and ART2 groups was significantly lower than those of VRT group (P ＜ 0.05 or 0.01), which nearly reached the levels of sham group (P ＞ 0.05).CONCLUSION: Androgen replacement therapy produces no significant effects on the morphological features of NOS-positive and nestin-positive neurons, but the therapy can selectively decrease the numbers of NOS-positive and nestin-positive neurons in different subregions of the basal forebrain, which may be closely related to androgen downregulation of expressions of NOS and nestin by ARs-mediated mechanisms, thereby producing complex effects on learning and memory processes.

Experimental basis for antioxidants TA99 series in the treatment of Alzheimer disease / 中国组织工程研究

OBJECTIVE: In recent years, available evidence from basic and clinical research on Alzheimer disease(AD) suggests that oxidation stress is involved in the occurrence and development of AD, and that antioxidant treatment can improve the intelligence of patients with AD and delay age-dependant cognitive dysfunction. Although results of basic and clinical research on the therapeutic effects of antioxidants on AD are inconsistent, a large number of available data suggest that these studies are of significance. Basic pharmacological studies on natural antioxidant TA99 series indicate that they are promising novel drugs for AD. Thereby, this study made a review of their experimental basis in the treatment of AD and existing problems.DATA SOURCES: Related articles published between January 1991 and December 2004 were searched by the computer in Medline database with such key words as Alzheimer disease, antioxidant, Ginkgo biloba extract, TA9901,acetylcholine, and senescence-accelerated mouse in different combinations and with the language limited to English. Meanwhile, related articles were alsosearched in CDMA \Wanfang database with the same key words in Chinese.STUDY SELECTION: Literature involving intervention group and control group were screened in the first trial, and then non-randomized trials were excluded and the rest were searched for the full text.DATA EXTRACTION: Of the 24 basic and clinical randomized and non-randomized trials on antioxidants in the treatment of AD collected, 17 accorded with the inclusion criteria and the other 7 were excluded.DATA SYNTHESIS: Intervention in the 17 trials emphasized the pathogenesis of AD from amyloid β proterin(Aβ) synthesis, gathering to senile plaque formation, and the enhancement of Aβ gathering and neuronal apoptosis by peroxidative injuries of free radicals. Both in vitro and in vivo studies were conducted: the effect of Aβ on neurons of different regions was observed with cell culture; transmission electromicroscope and sulfrin T (Th-T) fluorescence assay, Fuliye-transform infrared(FT-IR) spectrum apparatus, electron magnetic resonance(EPR), and round spectrum were used to detect the inhibitory effect of TA99 series on Aβ gathering and fibroplasia in vitro, as well as the influence on Aβ gathering in vivo. Senescence accelerated mouse (SAM) -P/8 was adopted to establish AD model and behavioral studies such as Morris water maze were used to investigate their effect on learning and memory. Meanwhile, the clearance of intracerebral amyloid granular deposition due to TA99 was also observed with hexamic argent staining. The effects of TA series on Aβ target and possible mechanism were fully revealed, and basic pre-clinical data collection was almost completed.CONCLUSION: TA9901 plant extractions have been proved to inhibit Aβ gathering and fibrosis, and improve learning and memory of SAM-P/8 rats. Moreover, TA9902 prepared by TA9901 combined with EGb761, another synergic herb, has an obvious anti-neurotoxic effect by inhibiting Aβ gathering, fibrosis and secondary structural changes. Further pharmacological research is needed and will have a promising prospect.

Protective effect of amyloid-beta 42 protein and its subunit vaccines immunization on spatial learning and memory of APPSWE transgenic mice / 中国组织工程研究

BACKGROUND: It has been demonstrated that amyloid-beta 42 protein (Aβ42) immunization in transgenic mouse models of Alzheimer disease(AD)can induce specific Aβ42 antibody, clear Aβ from the brain, and thereby improve spatial learning and memory. It has been a promising treatment strategy for AD.OBJECTIVE: To explore the effect of Aβ42 and its subunit vaccines immunization on spatial learning and memory of APPSWE transgenic mice.DESIGN: A randomized controlled experiment with mice as subjects.SETTING: The brain research laboratory of the anatomy department in a the medical college of a univeristy.MATERIALS: The experiment was conducted in the Experimental Animal Center and the Anatomy Department of Sun Yat-sen University from April 2003 to February 2004. Thirty-two APPSWE transgenic mice of 5 months old were bought from Taconic Company, USA. The second generation of mice were successfully reproduced in the Anatomy Department. These mice were randomly assigned into four groups: control group, Aβ42 group, Aβ1-15group, and Aβ36-42 group. Each group contained 8 in each group.INTERVENTIONS: Aβ42 and its subunits combined with MF59 adjuvant were subcutaneously injected for fundamental immunity and then applied in nasal mucosa for intensified immunization. The immunization period was 8 months. Y-maze was used for behavior test before immunization and Morris water maze was used after immunization.MAIN OUTCOME MEASURES: Spatial learning and memory, mean escape latency, times of passing through the platform point, swimming distance percentage of the first quadrant, and swimming distance percentage of the 20% marginal area.RESULTS: The correct reaction times in Y-maze behavior test were 7.50 ±0. 81, 7.06 ±0.71, 7.19 ±0.91, and 7.50 ±0.86 respectively in the control, Aβ42, Aβ1-15, Aβ36-42 groups and there was no significant difference ( P ＞ 0. 05) . After immunization, the mean escape latencies in 8 units of localized navigation test were(67.3 ±2. 8) s, (23.6 ± 1.6) s, (26.4 ±2.0) s,and (36.5 ± 2.2) s. The results in three experiment groups were different from that in control group and there was no difference between the three experiment groups ( P ＞ 0. 05 ) . The mean times of passing through the platform point in the 4 groups were 0.71 ±0.29, 8.14 ± 1.37, 7.28 ± 1.34,and 3.29 ± 0. 67. Swimming distance percentage of the first quadrant in the4 groups were(24.3 ±2.9)%, (50.6±11.6)%, (49.9±9.3) %,and(35.4±7.0)% and the swimming distance percentages of 20%marginal area were (46.4 ± 7.3 ) %, ( 11.6 ± 3.9) %, ( 14.4 ± 2. 6) %, and (25.8 ± 3.3)%. The mice in three experiment groups showed increase in the times of passing through platform point, swimming distance percentage of the first quadrant, and decrease in distance percentage of 20% marginal area compared with control group. The results in three experiment groups were no significantly different( P ＜ 0. 05).CONCLUSION: Immunization with A342 and its subunits can effectively ameliorate impairment of spatial learning and memory in APPSWE transgenic mice.

The comparative study of protective effect of two kinds of natural antioxidants on spinal motoneurons following brachial roots avulsion / 中华显微外科杂志

Objective To compare the effect of natural antioxidants TA9001 and EGb761 on c-jun, NOS expression and survival of spinal motoneurons following brachial roots avulsion. Methods One hundred and eighty adult Sprague-Dawley female rats were randomly divided into TA9001, ECb761 and control groups. The right C 5～T 1 nerve roots were avulsed and then the introperitoneal injection of 1ml of 0.5% TA9001, 0.5% EGb761 or normal saline was given immediately and once daily to the rats, respectively. The rats were killed after survival for 4 h, 12 h 1 d,3 d,5 d,and 1 week, 2 weeks, 4 weeks and 6 weeks. The cryostat sections of C 7 segment were prepared and carried with c-jun immunocytochemistry, NADPH-d histochemistry and neutral red counter stain. Results The c-jun and nNOS gene expression was only appeared in injured motoneurons. c-jun was first appeared at 4 h, reached its maximum at 1 d, and grandually decreased till 2 weeks. NOS was first checked at 5 d, mostly at 2 weeks and decreased until 6 weeks. Avulsed motoneuron death started at 2 weeks, reached its peak at 4～6 weeks. Both TA9001 and EGb761 can cause c-jun up-regulation, nNOS down-regulation and more motoneuron survival as compared to control. Furthermore, EGb761 had more power to enhance c-jun expression than TA9901 at each time point. Conclusion It seems that nNOS is more important in motoneuron death mechanism than c-jun. Treatment of either TA9901 or EGb761 can protect the injured motoneurons following root avulsion.

Ginkgo biloba extract enhances c-jun expression and attenuates motoneuron death induced by root avulsion / 中国病理生理杂志

AIM: To investigate the influence of Ginkgo biloba extract (EGb761) on c-jun expressions and motoneurons survival following root avulsion. METHODS: One hundred and eighty adult Sprague-Dawley female rats were randomly divided into control and EGb761 groups. Immediately after avulsion of C5-T1 nerve roots, the rats were injected ip with either 1 mL of EGb761 25 mg?kg~(-1)?d~(-1) or the same volume of normal saline, and the treatment repeated everyday. At 4 h to 6 weeks following avulsion, the C7 spinal segments of all rats were collected and prepared for c-jun immunocytochemistry and neutral red stain. The numbers of (c-jun) positive and survival motoneurons were counted and compared between two groups at each time point. RESULTS: In control rats following avulsion, c-jun positive motoneurons appeared at 4 h, reached its maximum at 1 d and declined to 2 weeks. Avulsion-induced motoneurons death started at 2 weeks, climbed to its maximum at 4 weeks-6 weeks. In EGb761 treated rats, both numbers of c-jun positive and survival motoneurons were more than that in control group at each time point. CONCLUSION: EGb761 attenuates avulsion-induced motoneurons death, and this effect may be related to up-regulation of c-jun gene in avulsed motoneurons. [

Influence of Ginkgo biloba extract on NOS expression and survival of C7 motoneurons following brachial root avulsion / 中国病理生理杂志

AIM: To investigate the influence of EGb761 on NOS expression and survival of motoneurons after spinal root avulsion.METHODS: Eighty adult SD female rats were randomly divided into control and EGb761 groups. The C5-T1 nerve roots of right brachial plexus were avulsed and injection of 1 mL of either EGb761 25 mg?kg -1 ?d -1 or normal saline (ip) was performed everyday. The treated rats were killed 1 week, 2 weeks, 4 weeks and 6 weeks following avulsion. The cryostat sections of C7 segment of every rat were collected and carried with NADPH-d histochemistry plus neutral red counterstain. The difference of the numbers of both NOS-positive and survival motoneurons were quantitatively studied. RESULTS: Following avulsion, NOS was expressed in avulsed motoneurons at 1 week, reached to its maximum at 2 weeks and then decreased gradually from 4 weeks to 6 weeks. Motoneurons died from 4 weeks to 6 weeks. With EGb761 treatment, the number of NOS-positive motoneurons were decreased at each time point and the number of survival motoneurons was increased at each time point compared to control rats.CONCLUSIONS: EGb761 protected the spinal cord motoneurons from avulsion injury. This effect may be related to inhibition of NOS expression.

A reactive pattern of ischemia-induced nestin protein in the rat brain / 中国病理生理杂志

AIM: To explore the expressive profile of nestin protein in the focal ischemic brain and to study the recovery mechanism of brain focal infarct . METHODS: Cellular morphology, time-course and distribution pattern of nestin positive response were immunohistochemically examined in different brain regions of 36 adult male SD rats. RESULTS: Nestin positive response of different brain regions in sham operated rats was present in small- and micro-vasculartures and the third ventricle bottom and ependyma. A large number of nestin positive cells were detected in ischemic brain, and were more remarkable in the cortical areas of parietal lobe and preoptic area as well as ischemic caudoputamen. Stellate nestin positive cells were located in the deep layer of ischemic cortex, but fibrillary cells were located in the shallow layer. Nestin positive cells in the ischemic caudoputamen showed the same changes of morphology as those cells in the deep layer of ischemic cortex. Morphological and number alterations of nestin positive cells were the most remarkable at 1 weeks post-ischemia, which showed more hypertrophy and proliferation in morphology, and a marked increase in number was present in the ischemic cerebral cortex and the ischemic caudoputamen. These alterations of nestin positive cells persisted up to 6 weeks post-ischemia, and then, the nestin positive response in the ischemic brain decreased gradually. CONCLUSION: Focal cerebral ischemia induces nestin re-expression on reactive astrocytes, which may be very important to the self-recovery of cerebral infarct.

Pathologic changes of spinal motoneurons function and morphology after root avulsion / 中国病理生理杂志

AIM: To investigate the pathological changes of motoneuron's function and morphology after root avulsion in order to study the neurobiology mechanism of motoneuron death. METHODS: Twenty female adult Sprague-Dawley rats, 200-300 g were used. The C 5-C 8, T 1 roots of the right brachial plexus were avulsed. All rats were killed 3 d, 5 d or 1 week after avulsion. One group of the C 5-C 8 spinal cords freeze sections (40 ?m thick) were collected for the NADPH-d histochemistry with neural red counterstained. Another group of the C 5-C 8 spinal cords freeze sections (40 ?m thick) were collected for the c-Jun immunocytochemistry stain. The paraffin sections (5 ?m thick) were collected for HE stain. The amount of NOS-positive and survival motoneurons was counted. The percentage of NOS expression and motoneuron survive were quantitatively analyzed considering the amount of contra lateral motoneurons as one hundred percent. RESULTS: The NOS expression rate was 0.74%?0.59% (3 d), 24.84%?6.73%(5 d), or 51.16%?8.67% (1 week), respectively. The survive rate was 93.00%?4.32% (3 d), 93.67%?5.27% (5 d), or 89.83%?2.65% (1 week), respectively. The motoneuron expressed c-Jun as early as 3 days after avulsion. The expression declined afterward until one week after avulsion. There was no significant change on the size of motoneuron. The nuclear membrane was still clear but some nuclei were not located in the middle of the cell body. There were some nucleoli with the chromatin condensation. CONCLUSION: The motoneuron NOS expression and cell death were increased within one week after spinal root avulsion. meanwhile, the c-jun expression was decreased. The NO/NOS may induce the motoneuron death by inhibiting the regenerating reactions of motoneuron after root avulsion injury.

Persistent response of microglia/macrophages following chronic focal cerebral ischemia in rats / 中国病理生理杂志

AIM: To study the microglial/macrophagic reactions to chronic foral cerebral ischmeia METHODS:ED1 and OX42 positive cellular reactive profiles including time-course and distribution as well as morphological changes were explored in the ischemic cortex and the ischemic caudoputamen of 36 SD adult rats by using focal cerebral ischemic model and immunohistochemical method RESULTS: On the 3rd day after ischemia, an increased number of round ED1 positive cells were found in the outer boundary of cortical ischemic foci and the ischemic caudoputamen, and some of the positive cells were present in the cortical ischemic core At 2nd week after ischemia, ED1 positive cells peaked in number, and they were located at cortical ischemic core and lateral caudoputamen, at which they persisted up to 6 weeks after ischemia. On the 3rd day after ischemia, ramified OX42 positive cells became hypertrophy and a marked increase in number, and they were present at the periphery of ischemic foci and in the ischemic caudoputamen At 2nd week after ischemia, OX42 positive cells became more hypertrophy, and a number of round OX42 positive cells were detected in the cortical ischemic core, in which they persisted up to 6 weeks after ischemia. CONCLUSION:Focal cerebral ischemia induces microglial/macrophagic reaction persistently, which may be correlative with neuronal delayed injury and self recovery of ischemic foci.

EFFECT OF TRANSPLANTATION OF THE NEURAL STEM CELLS MODIFIED WITH GENE OF NGF OR GDNF ON THE CHOLINERGIC NEURONS OF RASAL FOREBRAIN OF AD MODEL RAT / 解剖学报

Objective To investigate the effect of a single or combined transplantation of the neural stem cells(NSCs) modified with gene of NGF or GDNF on the cholinergic neurons of basal forebrain of AD model rat. Methods The NSCs modified with gene of NGF or GDNF were implanted in single or combined into the lateral cerebral ventricle of the rats after fibria\|fornix transection.The rats were killed three weeks after transplantation and the brain sections concluding basal forebrain were cut coronally on a freezing microtome and were processed by immunohistochemistry staining with antibodies against ChAT.The numbers of ChAT positive neurons of medial septum(MS) and vertical diagonal band(VDB) were analyzied statistically with one way of Student\|Newman Kaels. Results In MS,the percentages of ChAT positive neurons at the lesion side to the intact side in NGF group was 81% which was significantly higher than that in the lesion group(34%),NSC group(36%) and GDNF group(50%), P 0^05). Conclusion\ The injury cholinergic neurons can be protected in different extent after a single or combined transplantation of the neural stem cells modified with gene of NGF or GDNF.Among these three groups,greater protection was found in NGF group and NGF+GDNF group,and lesser protection in GDNF group.\;[