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.
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.
Objective Neuron purification is essential to procedure of various nerve cell experimental research, however, at present there is few reports on the effect of various factors on neural axons during purification. To find out a simple method of neuron purification, and to investigate the influence factors of corresponding purification culture in dorsal root gangl ion (DRG) tissue culture on β3-tubul in positive axon. Methods The DRGs were obtained from the 3 days neonatal SD rat microscopically and were made into cell suspension. Then, the amount of attached DRG neurons and non neuronal cells in poly-D-lysine (PDL) group, PDL/Laminin (PDL/LN) group and collagen-I (Col I) group was observed from 10 to 100 minutes. Then, the extension and arborization of β3-tubul in positive axons were observed after 72 hours completely randomised DRG tissue culture for the research of the influences among culture substrates (PDL, PDL/LN, and Col I), FBS (0, 5%, and 10%), 5 fluorouracil (5-Fu, 0, 20, and 40 μmol/L), and cytrarabine (Ara-C, 0, 10, and 20 μmol/L). Results Adherent cells were observed instantly after inoculation by inverted phase contrast microscope and inverted fluoresence microscope; after cell suspension was removed, adherent growth of DRGn cells and non-DRGn cells were still seen. In PDL group, the amount of NSE negative cells was significantly higher than that of NSE positive cells at 10 and 30 minutes (P lt; 0.05); the amount of NSE positive cells was significantly higher than that of NSE negative cells at 80, 90 and 100 minutes (P lt; 0.05). In PDL/LN gruop, there was no significant difference (P gt; 0.05) in the amount of NSE negative cells and NSE positive cells at 10, 20, 30, 40, and 50 minutes; the amount of NSE positive cells was significantly higher (P lt; 0.05) than that of NSE negative cells at 60, 70, 80, 90, 100 minutes. In Col I group, the amount of NSE negative cells was higher than that of NSE positive cells at 10-40 minutes, but showing no significant difference (P gt; 0.05); the amount of NSE positive cells was significantly higher (P lt; 0.05) than that of NSE negative cells at 70-100 minutes. At 72 hours after DRG tissue culture, the best result of β3-tubul in positive axon extension and arborization was obtained when the substrate level was PDL/LN, and the average length of PDL/LN level was significantly larger than that of other two substrates (P lt; 0.05). The highest number of β3-tubul in positive axon distal end was obtained at 5% concentration level of FBS (P lt; 0.05), but showing no significant differences in β3-tubul in positive axon length among three levels (P gt; 0.05). Both the most of β3-tubul in positive axon distal ends and the longest β3-tubul in positive axon average length were obtained at 0 μmol/L concentration level of 5-Fu, showing significant differences between 0 μmol/L level and 20, 40 μmol/L levels (P lt; 0.05). A similar result of β3-tubul in positive axon distal end was got at the 0 μmol/L level and 10 μmol/L level of Ara-C, which was significantly higher than that of 20 μmol/L level (Plt; 0.05). Conclusion? A purified DRG neuron suspension for neuron culture could be obtained via PDL differential attachment for 30 minutes. When DRG neuron culture, neuron special medium, PDL/LN substrate and 10 μmol/L Ara-C are recommended in β3-tubul in positive axon research.
Objective To investigate the effect of olfactory ensheathing cell culture medium (OECCM) on the growth of spinal cord neurons and its protective effect on the injured neurons by H2O2, and to disscuss the probable protective mechanisms of olfactory ensheathing cells (OECs). Methods The primary olfactory ensheathing cells (OECs) were isolated from olfactory bulb of adult SD rat, and OECCM were prepared. The morphology of OECs was observed by inverted phase contrast microscope, identified by rabbit-antiratlow-affinity nerve growth factor p75 (NGFRp75), and its purity were calculated.Primary spinal cord neurons were cultured from 15 to 17 days pregnant SD rats, and injury model of neurons were prepared by H2O2. OECCM and control culture medium were added into the normal spinal neurons (groups A, B). OECCM and control culture medium were added into the injured spinal neurons by H2O2 (groups C, D). In groups A and C, 200 μL of control culture medium was used; in groups B and D, 100 μL of control culture medium and 100 μL of OECCM were used. Then the growth index such as average diameter of neuron body, the number and length of neuron axons were measured. The viabil ities of normal and injured neurons were assessed by MTT. Results OECs showed bipolar or tripolar after 6-9 days of culture. Primary spinal cord neurons were round and bigger, and neuron axons grew significantly and showed bipolar after 5-7 days of culture. The immunocytochemisty of OECs by NGFRp75 showed that membrane were stained. The degree of purity was more than 90%. Primary spinal cord neurons grew well after 6-9 days of culture, and compared with group A, neurons of group B grew b, whose cell density and diameter were bigger. The average diameter of neuron body, the number and length of neuron axons were (33.38 ± 6.80) D/μm, (1.67 ± 0.80), and (91.19 ± 62.64) L/μm in group A, and (37.39 ± 7.28) D/μm, (1.76 ± 0.82), and (121.33 ± 81.13) L/μm in group B; showing statistically significant differences (P lt; 0.05). The absorbency (A) value of neurons was 0.402 0 ± 0.586 9 in group A and 0.466 0 ± 0.479 0 in group B; showing statistically significant difference (P lt; 0.01). After 2 hours of injury by H2O2, the cell density of spinal cord neurons decreased, and neuron axons shortened. The A value of injured neurons was 0.149 0 ± 0.030 0 in group C and 0.184 0 ± 0.052 0 in group D, showing statistically significant difference (P lt; 0.01). Conclusion The results above suggest that OECCM could improve the growth of spinal cord neurons and protectthe injured neurons. The neurotrophic factors that OECs secrete play an important role in the treatment of spinal cord injury.
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 research the protective effects of different allogeneic cells injected into denervated muscles on ventricornual motor neuron. Methods Thirty-six adult female SD rats, weighting 120-150 g, were individed into four groups randomly and each group had nine. Left ischiadic nerves of all the SD rats, which were cut down on germfree conditions,were operated by primary suture of epineurium. Different cells were injected into the triceps muscles of calf in each group after operation with once a week for 4 weeks:1 ml Schwann cells (1×106/ml) in group A, 1 ml mixed cells ofSchwann cells and myoblast cells (1∶1,1×106/ml) in group B, 1 ml extract from the mixed cells of Schwann cells, myoblast cells and endotheliocytes (1∶1∶1,1×106/ml)in group C,and 1 ml culture medium without FCS as control group(group D). The observation of enzymohistochemistry and C-Jun expression in the ventricornual motor neuron was made after three months of operation. Results After 3 months of operation, the expressions of C-Jun in groups A, B and C were superiorto that in group D; the number of neuron was more than that of group D. The expressions of C-Jun in the ventricornual motor neuron were as follows: 128.591±0.766 in group A, 116.729±0.778 in group B, 100.071±2.017 in group C and 144.648±2.083 in group D; showing statistically significant difference between groupsA, B, C and D(P＜0.01). Enzymohistochemistry showed the well outlined and wellstacked cell body of neuron in groups A, B and C, and illdefined boundary of cytoplasm and nucleus. There was statistically significant defference in enzyme activity of the ventricornual motor neuron between groups(P＜0.01). Conclusion All of the Schwann cells,mixed cells of Schwann cells with myoblast cells,and the extract from Schwann cells, myoblast cells and endotheliocytes can protect the ventricornual motor neuron. And the protectiveeffect of the extract from Schwann cells, myoblast cells and endotheliocytes is superior to that of Schwann cells and mixed cells.
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.
Objective To investigate the survival effect and reaction mechanismsof motor neurons after reimplantation of the avulsed root into the spinal cord,and to observe the survival and differentiation in the spinal cord after brachial plexus roots avulsion. Methods Thirty adult Wistar rats were randomly devided into the control group and the experimental group (n=15). Laminectomy of C4-6 was performed via a posterior approach. The ventral and dorsal roots of C5，6 were both avulsed from the spinal cord outside the dura mater and within the vertebral canal.For the experimental group, the ventral root of C6 wasreimplanted into the ventralhorn under microscope. The dorsal root was left. The ventral and dorsal roots of C5 were placed inside the nearby muscles. For the control group, the ventral and dorsal roots of both C5 and C6 were placed inside the nearby muscles. At 2, 4, 6, 8, 12 weeks postoperatively, the C6 spinal cord was stained with HE. The changes of the number and morphology of motor neurons were observed onHEstained sections. The C6 spinal nerve root was stained with silver nitrate, andthe regeneration of nerve fiber was observed. Results All rats were recovered well and their wounds were healed at primary stage. The gross observation showed that the avulsed nerve roots in control group adhered to adjacent muscles, however the one in experimental groups which had been implanted into spinal cord adhered to scar tissues and were not separated from spinal cord. At each time point postoperatively, the HEstained transverse sections showed that the number of motor neurons decreased significantly with soma swollen and atrophied, Nissle bodies decreased or disappeared. The survival rates of motor neurons in the control group were 60.9％±5.8%,42.3％±3.5％,30.6％±6.1％27.5％±7.9％ and 20.4％±6.8％ respectively；in the experimental group,the survival rates were 67.1％±7.4％,56.3％±4.6％,48.7％±8.8％,44.2％±5.5％ and 42.5％±8.3％ respectively. The survival rates of motor neurons in the experimental group was higher than those in the control group at all time points,showing statistically significant difference（Plt;0.01）.At 12 weeks postoperatively, thesilver nitrate stained specimen from the C6 nerve root showed regeneration of the motor neurons in the ventral horn into the reimplanted nerve root through axon in the experimental group,but the degeneration of the nerve fiber appeared and the number of the myelinated nerve fiber decreased in the control group. Conclusion Through reimplantationof the avulsed ventral nerve root into the ventral horn, degeneration of the motor neurons in the ventral horn can be reduced. After reimplantation of avulsed nerve root, there is axonal regrowth of motor neurons into the spinal nerve root and regeneration of the myelinated nerve fiber also appears.
Objective To investigate the distribution of rats’ pelvic muscles motoneurons innervated by artifical somatic-autonomic reflex arc. Methods Thirty-five SD rats were randomly divided into normal group (n=10) and model group (n=25). The rats in the normal group were given no treatment. In the normal group, the artifical somatic-autonomic reflex arc was established. Six months after establishing the model, external urethral sphincter (EUS), ischiocavernosus (IC), bulbocavernosus (BS) and external anal sphincter (EAS) of the rats in normal group(n=10) and of the rats in model group A (n=20) were injected with fluorogold (FG). The reversal neural tracing was done. FG positive neural cells were observedby fluorescent microscope. Malt agglutinator binding horseradish peroxidase (WGA-HRP) was injected into L4 spinal cord of the rats in model group B (n=5) as the anterograde tracer. After being treated with TMB-HRP reaction, the axon endings in the neuromuscular junction in pelvic striated muscles (EUS, IC, BS, EAS) were investigated with light microscopes. Results In normal group, EUS and IC injections resulted in transport of FG to neurons in the dorsolateral nucleus (DL) of the ventral horn of the L5～S1, and BS and EAS in the dorsomedial nucleus (DM) of ventral horn in the L5～S1. In the model group A, EUS, IC, BS andEAS injections resulted in transport of FG to neurons in the left ventral horn in the L4. In model group B, after WGA-HRP was injected into the L4 left ventral horn, HRP positive axon terminals were observed in the EUS, IC, BS and EAS. Conclusion In the normal rats, the pelvic striated muscles motoneurons locate in the ventral horn of L5～S1. In the model rats, the pelvic striated muscles motoneurons innervated by artificial somatic-autonomic reflex arc locate in the ventral horn of the L4. After the artificial somaticautonomic reflex arc is established, the isomerous nerve fiber innervates EUS, IC, BS and EAS.
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.