Objective Targeted adenoviral gene delivery from peripheral nerves was used to integrally analyse the characterization and time course of LacZ gene (AdLacZ) retrograde transfer to spinal cord and transgene product anterograde labeling ofperipheral nerve. Methods Recombinant replication-defective adenovirus containing AdLacZ was administrated to the cut proximal stumps of median and tibial nerves in Wister rats. Then the transected nerve was repaired with 10-0 nylon sutures. At different time point postinfection the spinal cords of C5 to T1 attached with DRGs and brachial plexuses, or L2 to L6 attached with DRGs and lumbosacralplexuses were removed. The removed spinal cord and DRGs were cut into 50 μm serialcoronal sections and processed for X-gal staining and immunohistochemical staining. The whole specimens of brachial or lumbosacral plexuses attaching with theirperipheral nerves were processed for X-gal staining. The number of X-gal stained neurons was counted and the initial detected time of retrograde labeling, peaktime and persisting period of gene expression in DRG sensory neurons, spinal cord motor neurons and peripheral nerves were studied. Results The gene transfer was specifically targeted to the particular segments of spinal cord andDRGs, and transgene expression was strictly unilaterally corresponding to the infected nerves. Within the same nerve models, the initial detected time of gene expression was earliest in DRG neurons, then in the motor neurons and latest in peripheral nerves. The persisting duration of β-gal staining was shortest in motor neurons, then in sensory neurons and longest in peripheral nerves. The initial detected time of β-gal staining in median nerve models was earlier in mediannerve models compared with that in the tibial nerve models. Although the initial detected time and the beginning of peak duration of β-gal staining were not same, the decreasing time of β-gal staining in motor and sensory neurons of thetwo nerve models were started at about the same day 8 post-infection. The labeled neurons were more in tibial nerve-models than that in median nerve models. Within the same models, the labeled sensory neurons of DRGs were morethan labeled motor neurons of ventral horn. The β-gal staining was tenser in median nerves than that in tibial nerves. However the persisting time of β-gal staining was longer in tibial nerve models. Conclusion The b gene expression in neurons and PNS renders this system particularly attractive for neuroanatomical tracing studies. Furthermore this gene delivery method allowing specific targeting of motor and sensory neurons without damaging the spinal cord might offer potentialities for the gene therapy of peripheral nerve injury.
摘要:目的:分析高膽紅素血癥新生兒血清神經元特異性烯醇化酶(NSE)含量和新生兒行為神經能力測評(Neonatal Behavioral Neurological Assessment,NBNA)的變化,探討高膽紅素血癥新生兒血清NSE含量變化的臨床意義。方法:應用放射免疫分析法分別測定60例高膽紅素血癥新生兒和20例對照組新生兒血清NSE含量,同步測定血清總膽紅素(TSB),進行NBNA評分;高膽紅素血癥組早期干預后再次測定血清NSE含量。結果: 與對照組比較,高膽紅素血癥新生兒血清TSB、NSE含量顯著升高,而NBNA評分明顯降低,差異有顯著性意義(Plt;0.01);對照組與高膽紅素血癥新生兒輕度增高、中度增高、重度增高四組兩兩比較(均Plt;0.05),存在顯著性差異;血清NSE含量與NBNA評分呈明顯負相關(r=-0628,Plt;0.01);高膽紅素血癥新生兒經早期干預治療后,血清NSE含量均下降(Plt;0.05),差異有顯著性。結論: 高膽紅素血癥可導致新生兒腦損傷,血清NSE含量可以作為腦損傷的監測指標。Abstract: Objective: To analyze levels of neuronspecific enolase(NSE)in serum and neonatal behavioral neurological assessment (NBNA), to study whether NSE in serum can be used as a tool for the early identification of brain damage in neonatal hyperbilirubinemia. Methods: Serum NSE level of 60 full term infants with hyperbilirubinemia and 20 cases as to control group were measured by radioimmunoassay; Also total serum bilirubin (TSB) and NBNA were detected. In the hyperbilirubinemia group,serum NSE level were measured second when TSB were less than 855 μmol/L(5 mg/dL). Results: Compared with control group,the levels of serum TSB、NSE of the hyperbilirubinemia group were significantly higher, but NBNA score was significantly lower. The levels of serum NSE was significantly negative related to NBNA score. In the hyperbilirubinemia group, serum NSE level were significantly lower after treatment. Conclusion: Hyperbilirubinemia in neonates can cause brain damage. Serum NSE level could work as monitoring indexes of this damage.
Repeated transcranial magnetic stimulation (rTMS) is one of the commonly used brain stimulation techniques. In order to investigate the effects of rTMS on the excitability of different types of neurons, this study is conducted to investigate the effects of rTMS on the cognitive function of mice and the excitability of hippocampal glutaminergic neurons and gamma-aminobutyric neurons from the perspective of electrophysiology. In this study, mice were randomly divided into glutaminergic control group, glutaminergic magnetic stimulation group, gamma-aminobutyric acid energy control group, and gamma-aminobutyric acid magnetic stimulation group. The four groups of mice were injected with adeno-associated virus to label two types of neurons and were implanted optical fiber. The stimulation groups received 14 days of stimulation and the control groups received 14 days of pseudo-stimulation. The fluorescence intensity of calcium ions in mice was recorded by optical fiber system. Behavioral experiments were conducted to explore the changes of cognitive function in mice. The patch-clamp system was used to detect the changes of neuronal action potential characteristics. The results showed that rTMS significantly improved the cognitive function of mice, increased the amplitude of calcium fluorescence of glutamergic neurons and gamma-aminobutyric neurons in the hippocampus, and enhanced the action potential related indexes of glutamergic neurons and gamma-aminobutyric neurons. The results suggest that rTMS can improve the cognitive ability of mice by enhancing the excitability of hippocampal glutaminergic neurons and gamma-aminobutyric neurons.
Objective To study the method to inhibit perioperative internal mammary artery (IMA) spasm from the perspective of muscarinic receptor, and research the function of muscarinic cholinergic receptor subtypes of IMA. Methods IMA segments in vitro with intact endothelium were obtained from 30 patients who underwent coronary artery bypass grafting (CABG). According to muscarinic receptor antagonists of different concentrations， They were divided into control group (not using receptor antagonist), atropine group (nonselective M receptor antagonist), pirenzepine group (M1 receptor antagonist) and Methoctramine group(M2 receptor antagonist) by random number table. The effects of antagonists on vasodilatation were analyzed, Scott ratio was used to calculate affinity index (pD2) and Schild plot was used to count rivalry index (pA2). Results Acetylcholine (Ach)induced concentrationdependentrelaxation response of IMA segments with intact endothelium precontracted with potassium chloride (KCl). The pD2 was 6.92±0.05. The effects of atropine, pirenzepine and methoctramine on doseresponse curve induced by Ach with intact endothelium were all concentrationdependent. With the increase of the concentration of antagonists, the Achinduced doseresponse curves had a significant shift to right(Plt;0.05). Atropine, pirenzepine and Methoctramine competitively antagonized the reaction of vessel to Ach. The pA2 were 9.62±0.15,7.70±0.08 and 630±0.08, respectively. Conclusion The Achinduced relaxation response of IMA with intact endothelium is concentrationdependent. According to the affinity of different antagonist, IMA in Vitro Achinduced relaxation response is implemented by acting on nonneuronal muscarinic cholinergic M1 receptor subtype.
ObjectiveTo observe the morphological and functional changes of retinal degeneration in mice with CLN7 neuronal ceroid-lipofuscinosis, and the therapeutic effects of glial cell derived neurotrophic factor (GDNF) and/or ciliary neurotrophic factor (CNTF) based on neural stem cells (NSC) on mouse photoreceptor cells. MethodsA total of 100 CLN7 mice aged 14 days were randomly divided into the experimental group and the control group, with 80 and 20 mice respectively. Twenty C57BL/6J mice aged 14 days were assigned as wild-type group (WT group). Mice in control group and WT group did not receive any interventions. At 2, 4, and 6 months of age, immunohistochemical staining was conducted to examine alterations in the distribution and quantity of cones, rod-bipolar cells, and cone-bipolar cells within the retinal of mice while electroretinography (ERG) examination was utilized to record scotopic a and b-waves and photopic b-wave amplitudes. At 14 days of age, the mice in the experimental group were intravitreally injected with 2 μl of CNTF-NSC, GDNF-NSC, and a 1:1 cell mixture of CNTF-NSC and GDNF-NSC (GDNF/CNTF-NSC). Those mice were then subdivided into the CNTF-NSC group, the GDNF-NSC group, and the GDNF/CNTF-NSC group accordingly. The contralateral eyes of the mice were injected with 2 μl of control NSC without neurotrophic factor (NTF) as their own control group. At 2 and 4 months of age, the rows of photoreceptor cells in mice was observed by immunohistochemical staining while ERG was performed to record amplitudes. At 4 months of age, the differentiation of grafted NSC and the expression of NTF were observed. Statistical comparisons between the groups were performed using a two-way ANOVA. ResultsCompared with WT group, the density of cones in the peripheral region of the control group at 2, 4 and 6 months of age (F=285.10), rod-bipolar cell density in central and peripheral retina (F=823.20, 346.20), cone-bipolar cell density (F=356.30, 210.60) and the scotopic amplitude of a and b waves (F=1 911.00, 387.10) in central and peripheral retina were significantly decreased, with statistical significance (P＜0.05). At the age of 4 and 6 months, the density of retinal cone cells (F=127.30) and b-wave photopic amplitude (F=51.13) in the control group were significantly decreased, and the difference was statistically significant (P＜0.05). Immunofluorescence microscopy showed that the NSC transplanted in the experimental group preferentially differentiated into astrocytes, and stably expressed CNTF and GDNF at high levels. Comparison of retinal photoreceptor nucleus lines in different treatment subgroups of the experimental group at different ages: CNTF-NSC group, at 2 months of age: the whole, central and peripheral regions were significantly different (F=31.73, 75.06, 75.06; P＜0.05); 4 months of age: The difference between the whole area and the peripheral region was statistically significant (F=12.27, 12.27; P＜0.05). GDNF/CNTF-NSC group, 2 and 4 months of age: the whole (F=27.26, 27.26) and the peripheral area (F=16.01, 13.55) were significantly different (P＜0.05). In GDNF-NSC group, there was no statistical significance at all in the whole, central and peripheral areas at different months of age (F=0.00, 0.01, 0.02; P＞0.05). ConclusionsCLN7 neuronal ceroid-lipofuscinosis mice exhibit progressively increasing degenerative alterations in photoreceptor cells and bipolar cells with age growing, aligning with both morphological and functional observations. Intravitreal administration of stem cell-based CNTF as well as GDNF/CNTF show therapeutic potential in rescuing photoreceptor cells. Nevertheless, the combined application of GDNF/CNTF-NSC do not demonstrate the anticipated synergistic protective effect. GDNF has no therapeutic effect on the retinal morphology and function in CLN7 neuronal ceroid-lipofuscinosis mice.
Objective To explore the changes of morphology and ventricornual motor neuronsin SD rats’ ventral horn of spinal cord after radiated as the therapy protocol for breast cancer, to discover the rule of radiationinduced injury of brachialplexus, and also if there exits the reversible conversion in neurons. Methods Twenty SD rats were selected. The left side of the rats was used as the radiation side, and the right side as the control side. The RIBPI animal models were established by divideddose of radiation. Using 2 Gy/time and 5 times/week, a total administered dose reached 30 Gy after 3 weeks. The behaviour of the rats was observed after radiation. At 3, 5, 7 and 9 weeks after the last radiation (n=4), the wet weights of biceps brachii muscle, upperlimb circumference and compound action potential were examined; the pathological changes of biceps brachiimuscle, the morphological changes, counts of the motor neurons in ventral horn and axons of bilateral spinal cord were observed by HE staining, argentums staining and toluidine blue staining. Results The rats showed lameness and a “claw hand” 3 weeks after radiation. Compared with control side, thewet weights of biceps brachii muscle and upperlimb circumference were significantly reduced, meanwhile, the compound action potential significantly decreased, and its latent period was also significantly prolonged 3, 5, 7 and 9 weeks (Plt;0.05). The histological observation: Musculocutaneous nerve showed decreased medullated fibers, heterogeneous ditribution and decreased density, thin myelin sheath, damaged nerve structure and collagen hyperplasia; biceps brachii muscle showed degeneration, fiber breakage and inflammatory cell infiltration; The account of motor neurons in ventral horn was significantly decreased in the radiation side with time extending, the sign of cell death, such as, the neurons crimple, and karyolysis were observed(Plt;0.05). Conclusion Large dose of X-ray can inducedbrachial plexus injury, and the lameness, a “claw hand”, biceps brachii muscle atrophy and the compound action potential abnormality. The account of motor neurons in ventral horn was significantly decreased. The motor neurons showed oxonal degeneration and myelinec degeration.
This research was to study the regulation of intravenous administration of human umbilical cord blood mesenchymal stem cells (HUCBMSCs) on secretion of neural specific protein in rats after traumatic brain injury (TBI), and to explore its mechanisms promoting the recovery of neurological function. The TBI models of rats were established. We then injected HUCBMSCs, labelled by Brdu (5-bromo-2-deoxyuridine), into the TBI rats via the tail vein using modified Feeney free-falling method. The levels of neural biochemical indicators (serum S100βprotein, NSE, LDH, CK) of rats were detected in shamed group, injury group and HUCBMSCs-transplanted group. And the morphological changes of brain tissue of rats in the three groups were observed by using HE staining under light microscope. During the whole experiment no immunosuppressant was used for the four groups. From the research, transplant-related death of the rats was not found in transplantation group. In the injury group, rises were found in contents of serum S100βprotein, NSE, LDH, CK in the early stage after the rats were injured, which were much higher than those in shamed group at correspondent time point(P < 0.01). In HUCBMSCs-transplanted group, although these biochemistry indexes were found rising for a short period in the early stage, along with the time, these indexes were obviously lower than in those injury group (P < 0.05). Under light microscopy pathological changes of rats in HUCBMSCs-transplanted group were much slighter than those in injury group. It was well concluded that in the situation of no immuno-suppressants, the intravenous-injected HUCBMSCs could reduce the secretion of serum S100βprotein, NSE, LDH, CK, promote the repair of tissue injury effectively, and promote the functional recovery of neurons.
OBJECTIVE: To purify and study Schwann cells cytoplasmic neurotrophic protein. METHODS: The dissociated SC taken from 300 newborn rats sciatic nerves were cultured, collected, ultrasonicated and ultraspeed centrifuged. The supernates were ultrafiltrated and concentrated by using ultrafiltration units with PM10, PM30, PM50 ultrafiltration membranes. The ultrafiltrated-concentrated solution with the protein molecular weight 10-30 ku, 30-50 ku and gt; 50 ku were collected respectively. The dissociated spinal cord motoneurons of 14 days embryonic rats were cultured with serum-free conditional medium and the additional SC cytoplasmic proteins were added into the medium. The results showed that the 10-30 ku and gt; 50 ku SC cytoplasmic proteins were able to maintain the survival of motoneurons for 24 hours. Then the 26 ku and 58 ku proteins were further extracted and purified from SC cytoplasm by high pressure liquid chromatography, and their neurobiological activities were studied. RESULTS: The 26 ku and 58 ku Schwann cell’s cytoplasmic proteins were able to maintain the survival of motoneurons cultured in the serum-free medium for 48 hours. The highest biological activity concentration is 20 ng per well. CONCLUSION: Schwann cells cytoplasm contains motoneuron neurotrophic proteins with molecular weight 26 ku and 58 ku.
Objective To evaluate the feasibil ity of direct anastomosis in the rat model of the brachial plexus extravertebral foramen nerve root division of C5-7. Methods Forty-eight SD rats (male or female) aging 4-6 months and weighing 250-300 g were selected to make the model of extravertebral foramen nerve root division of C5-7. The left C5-7 nerve roots, as the experimental sides, were separated to the brachial plexus nerve trunk and the transected roots were sutured to theproximal stump immediately after cutting off the brachial plexus extravertebral foramen nerve root division. The right C5-7nerve roots, as the control sides, received no operation. The general condition of rats after operation was observed. The gross observation, the histological observation and BDA nerve tracing technology were adopted to observe the wet weight of musculus biceps brachii, the cross section of biceps brachii and the spinal cord and distal nerve trunk at 3 weeks, 3 months and 6 months after operation. Results All rats survived well after operation. Claudication and unfold claw reflex were observed in the experimental sides, and the unfold claw reflex disappeared 3 months later. Comparatively, the control sides were normal. Nerve adhesion aggravated gradually and the neural stems were shriveled within 6 months after operation in the experimental sides. Comparatively, the control sides were normal. The wet weight of biceps brachii in the experimental sides and the control sides at 3 weeks, 3 months and 6 months after operation was (0.28 ± 0.12), (1.37 ± 0.33), (0.58 ± 0.10), (1.36 ± 0.35), (1.39 ± 0.31), (1.37 ± 0.38) g, respectively, indicating significant differences between two sides at 3 weeks and 3 months (P lt; 0.05), but no significant difference at 6 months (P gt; 0.05). The modified Marsland and the LFB staining of spinal cord and superior trunk of brachial plexus showed that the number of neurons, cell nuclear and Nissl body decreased and cell bodies changed from swell ing to shrinkage, dyeing nerve fibers increased, neural axone was thin and myel in sheath was sl ightly stained at each time point in experimental side. The number of motor neurons in cornu anterius medullae spinal is in the experimental side was 84.5% ± 3.2%, 74.4% ± 4.5%, 73.7% ± 3.8% of that in the control side at each time point, respectively. HE staining of biceps brachii detected thatthe muscle denaturation was very serious at 3 months after operation and then recovered. Neural tracing used BDA showed that the closer to the proximal of nerve trunk, the more obviously stained it was of myel in sheath and the more massive of axon at 6 months after operation. And there was almost no myel in and axon stained in musculocutaneous nerve. Conclusion In the rat model of brachial plexus extravertebral foramen nerve root division, the motoneuron in cornu anterius medullae spinal is necrosis rate reaches 20%-30%, and most of the residual neurons are pathologic. The regenerated fibers manifest as insufficient dynamic power and incomplete development, making no sense for the recovery of end organ function. Therefore, the exact mechanism of the recovery of biceps brachial muscle demands further study.
Objective To summarize the clinical features of motor neuron disease ( MND) with main presentation of pulmonary hypertension, so as to improve the diagnosis.Methods A patientwithMND whose main presentation was pulmonary hypertension was analyzed retrospectively. Meanwhile related literatures were reviewed. Clinical data including symptoms, early signs, misdiagnosis causes, and necessary functional examination of respiratory muscle were collected. Results The symptoms of MND was slow-onset and insidious with gradual progression over time. History inquiring found that the symptoms of muscle wasting and physical debilitation emerged long time before the respiratory symptoms. Physical examination also revealed obvious sign of muscle atrophy. Conclusions MND with main presentation of pulmonary hypertension has been recognized insufficiently and often misdiagnosed as other pulmonary diseases. Detailed history taking, systematic physical examination, and convenient functional examination of respiratory muscle,can not only reduce misdiagnosis, but also avoid some expensive and traumatic process.
