| BackgroundCurrently with the developing of the biological information technology, the means of monitoring brainstem function are improved and are diversified gradually, including BAEP, SEP and MRI and so on. But as bedside diagnostic monitoring tools, they still had some limitations. Therefore, the study will investigate a clinical bedside monitoring method which can be operated non-invasively and simply. It can be used to help the clinician to judge the brainstem function lesion objectively and accurately. The study will construct a foundation for its further clinical application.Purposes1 Through constructing the experimental basal ganglia intracerebral hemorrhage model in rats induced by different doses of collagnase, to observe the changes of intracranial pressure, pathological changes of different brainstem segments, spontaneous nystagmus and ice water-induced ENG and the relationships among them. It was achieved that the feasibility of the ENG being applied to monitor the function of brainstem dynamicly.2 Through observing the changes of nucleus and the medial longitudinal fasciculi in brainstem and the relationships with ICP, the pathological bases were explored how the spontaneous nystagmus and the abnormal nystagmus induced by ice water test occurred after the experimental basal ganglia intracerebral hemorrhage model in rats induced by different doses of collagnase.3 To further verificate brainstem damage being the cause of spontaneous nystagmus and the abnormal ENG ice water-induced after the experimental basal ganglia intracerebral hemorrhage, the similarities and differences of spontaneous nystagmus and ice water-induced ENG were compared between the rat models of which the brainstems were transected at different levels and of the basal ganglia intracerebral hemorrhage. It could provide evidences for ENG being applied to judge the brainstem damage after ICH.4 To explore the mechanisms of spontaneous nystagmus and the abnormal ENG ice water-induced caused by brainstem damage, the changes of spontaneous nystagmus and ENG ice water-induced were observed after the brainstem nucleis and the medial longitudinal fasciculus in different stages being destructed and stimulated respectively. It could provide evidences for ENG being applied to judge the brainstem damage after ICH. MethodsPart I: The value which ice water-induced ENG was used being the method to judge the brainstem damage after the experimental basal ganglia intracerebral hemorrhage in rats.1 The experimental basal ganglia intracerebral hemorrhage (ICH) was set up according with the Rosenberg's way. Rats were divided into five groups: sham group, saline control group, 0.3U collagenase injection group, 0.6U collagenase injection group, 0.9U collagenase injection group.2 Electrophysiological recording: RM6240C multi-channel physiol- ogical recorder recorded the curve of the intracranial pressure, the spontaneous nystagmus and the ENG ice water-induced at 12h, 24h, 48h, 72h, 5d and 7d after ICH.3 Pathological changes of brainstem was observed through HE staining.4 Intracranial pressure, pathological changes of the different brainstem segments and ENG ice water-induced including latency (Lat), frequency (F), amplitude (Amp), slow-phase velocity (SPV), circadian variation index (CVI) were given correlation analysis. The function was detectived that the parameters of ENG ice water-induced was applied to judge the different segments of brainstem damage through discriminant analysis.Part II: The occurring mechanism of the spontaneous nystagmus and the abnormal ice water-induced ENG after the experimental basal ganglia intracerebral hemorrhage in rats.1 The expression levels of NMDAR1 and GABA-ARα1 in oculomotor nucleus (OMN) were detected by immunohistochemistry being responsed to the changes of neuron in the molecular level. TUNEL staining determined the degrees of apoptosis at different brainstem segments being responsed to the changes of neuron in cellular level. Immunohistochemical method measured the MBP expression level of medial longitudinal fasciculus in the brainstem at different layers being responsed to the changes of brainstem myelin.2 The changes of spontaneous nystagmus and ENG ice water-induced after the different layers of brainstem were transeced in rats. The brainstem was exposed fully through the enter way of the cerebellomedullary choroidal fissure which the cerebellum was intact. The brainstem was transected at different layers: superior to midbrain quadrigeminal body, inferior to midbrain quadrigeminal body, superior pons, inferior pons. Changes of respiration, blood pressure, spontaneous nystagmus and ENG ice water-induced were also recorded by RM6240C multi-channel physiological recorder.3 The changes of spontaneous nystagmus and ENG ice water-induced after the different nucleis and the different stages of the medial longitudinal fasciculus were electric coagulated damaged and electrical stimulated. One lateral oculomotor nucleus, paramedian pontine reticular nucleus and vestibular nucleus were damaged by electrocoagulation and electric stimulated respectively. The different stages of medial longitudinal fasciculus were electrocoagulated at the layer of superior midbrain quadrigeminal body, the layers of superior and inferior midbrain quadrigeminal body, the layers of superior and inferior midbrain quadrigeminal body and superior pons, the layers of superior and inferior midbrain quadrigeminal body, superior pons and inferior pons. Spontaneous nystagmus and parameters of ENG ice water-induced were analyzed and compared among the different nucleis being damaged or stimulated, so as to the different layers of the medial longitudinal fasciculus being damaged.ResultsPart I:1 The level of ICP was normal in 0.3U collagenase injected group at 12h, rose at 24h and reached the climax at 48h-72h. ICP of 0.6U and 0.9U collagenase injected group were heightened at 12h and reached the peak at 48h-72h (25mmHg). The more the collagenase were injected the ICP were higher at same time.2 Different style spontaneous nustagmus were recorded at different period after ICH. There had significant difference between ICH groups and control group in the parameters of ENG ice water-induced at each time (P<0.01), except for 0.3U collagenase injected group at 12h (P>0.05). The more the dosage of collagenase was injected, the sooner the changes of ice water induced nystagmus occured, the longer the latency was, the slower the frequency was, the smaller the amplitude was, the slower the slow-phase velocity was, the greater circadian variation index was (P<0.01).3 Pathological changes of brainstem: HE staining showed that the neurons being swelling , the nucleus being condensed and deep-dyed and the neuronal fibers structurel being disordered. The accumulation of inflammatory cells and bleeding also could be seen. The higher dose of collagenase being injected, the higher ICP elevated, the more obviously the pathological changes occurred in brainstem. The developing sequence was from up to down within brainstem.4 The accurate rate to judge the brainstem lesion localization with parameters of ENG ice water-induced including Lat, F, Amp, SPV, CVI could reach 79.5% through discriminant analysis.Part II:1①The NMDAR1 expression levels in OMN of ICH model groups were less than that of control group (P<0.01) at each time. The more the dosage of the collagenase injected, the lower its expression level was and the expression level of GABA-ARα1 had no difference (P>0.05) compared with that of control group.②The percentage of positive apoptotic cells within the brainstem in all ICH groups were higher than that of the control groups (P<0.01). The apoptotic cells were located in the upper layers of the midbrain or pons at 12h-24h after ICH. Then percentage of positive apoptotic cells at all layers of brainstem increased at 48h-72h (P<0.01). The percentage of positive apoptotic cells at inferior pons were increased in 0.6U and 0.9U collagenase injection groups (P<0.01). Comparing among the ICH groups at the same period, the greater the dose of collagenase being injected, the higher the percentage of apoptotic cells was (P<0.05). The results suggested that the greater the doses of collagenase being injected, the earlier the cell apoptosis of brainstem occured, the higher the percentage of apoptotic cells was and the larger the range of apoptosis was involved. The developing sequence was from the midbrain to inferior pons.③It could be seen that coloring of myelin being shallow which bundle structure was disrupted and the average optical density decreased in all ICH groups (P<0.01). It indicated the presence of demyelination. The average optical density of MBP of medial longitudinal fasciculus in the brainstem declined at 72h, 24h, 12h after 0.3U, 0.6U, 0.9U collagenase being injected respectively. The trend showed degrading over time and dropped to the lowest point at 48h-72h compared with the control group (P<0.01). The average optical density descending of MBP in upper pons appeared in 0.6U and 0.9U group at 72h and 48h respectively. There had significant difference within groups (P<0.01). Abnormal expression of MBP in inferior pons only was showed in 0.9U group at 48h (P<0.05), which showed the abnormal expression of MBP of the whole brainstem MLF from the midbrain to pontine. The higher dose of collagenase being injected, the smaller the average optical density of the MBP was, the more obviously the demyelination occurred. The developing sequence was from up to down within brainstem also. Correlation analysis showed that the higher the intracranial pressure was, the lower level of NMDAR1 in the OMN expressed, the lower level of MBP of MLF expressed and the higher level of cell apoptosis within brainstem was.2 Different types of spontaneous nystagmus appeared after the different brainstem segments being transected. The types of spontaneous nystagmus were upbeat nystagmus or torsion spasm nystagmus after the superior midbrain quadrigeminal body being transected. The types of spontaneous nystagmus were downbeat nystagmus after the superior pons being transected. The types of spontaneous nystagmus were eye snap or periodic alternating nystagmus after the inferior pons being transected. All parameters of ENG ice water-induced after midbrain transection had no significant difference compared with the control group (P>0.05). Nystagmus couldn't be induced by ice water after other brainstem segments being transected.3①There was no difference at Lat and F of ENG ice water-induced between the group which the right lateral OMN being damaged and control group (P>0.05), while the Amp and the SPV descended, the CVI increased (P<0.05). The parameters of ENG ice water-induced while the right OMN being stimulated had no difference compared with the control group (P>0.05).②There was no difference in the type of ENG left lateral ice water-induced after the right PPRN being damaged compared with the control group, but the Lat extended, the F and Amp were down, the SPV was slow, the CVI increased (P<0.05). Nystagmus couldn't be induced by right ice water test. There was no difference in the type of ENG right lateral ice water-induced after the right PPRN being stimulated compared with the control group, same as the Lat, while the F, the Amp, the SPV and the CVI increased (P<0.05). While the left water induced ENG showed the F, the Amp, the SPV decresed and the CVI was greater(P<0.05).③There was no difference in the type of ENG right lateral ice water-induced after the right VN being damaged compared with the control group, same as the Lat, the Amp, the SPV and the CVI increased (P<0.05). Nystagmus couldn't be induced by left lateral ice water test. There was no difference in the type of ENG left lateral ice water-induced after the right VN being stimulated compared with the control group , same as the Lat, while the F, the Amp, the SPV and the CVI increased (P<0.05).④After the MLF of superior midbrain quadrigeminal body being electric coagulated, the Lat, F, Amp, SPV and the CVI of the ENG ice water-induced were same as those of the control group (P>0.05). Along with the more layers being destructed from superior to inferior, the latency was extened, the frequency and slow phase velocity became slower, the amplitude descended and the CVI increased about the ENG ice water-induced (P<0.05). The nystagmus induced by ice water test disappeared after the whole MLF of brainstem being damaged.ConclusionsSpontaneous nystagmus and nystagmus ice water-induced could occur different changes at different periods after the basal ganglia ICH model in rats. The reasons were the damages of the nucleus and MLF about the eye movements in brainstem caused by the different degree increased ICP. On the contrary, the locations and the degrees of brainstem damage could be judged according with parameters of ENG ice water-induced and spontaneous nystagmus...
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