| BackgroundCerebrovascular disease is the leading death cause of urban residents in China. Intracranial hemorrhage (intracerebral hemorrhage, ICH) accounts for about 15% of stroke patients, with the mortality about 50% within the first month after attack. The incidence age has become much younger in recent years. The ICH bleeding location of 5-10% of patients is in the cerebellum with the final mortality rate as high as 20-30%. Autopsy studies showed that death of cerebellar hemorrhage was mainly caused by herniation by which the brainstem was oppressed and destructed under the circumstance of the hemorrhage and edema. Therefore, it was critical to monitor the changes of patients'condition and apply early interventional strategy to treated the disease and save lives. At present, CT is the most reliable detecting method for the diagnosis of the cerebellar hemorrhage. However, CT is not a dynamic diagnostic method, whereas the repeated exam to observe the changing condition of the patients increase the the costs. Another disadvantage is the movement of the patients may worse the condition of the patients. A method which can be operated bedside in time is largely needed to monitor the progression of the patients'condition which to discriminate whether the brainstem was intact. The study was to find a clinical useful monitoring method which has the characteristics including bedside, dynamic, non-invasive, simple, objective, accurate. Importantly, it is that it can be applied to anticipate the occurrence of brainstem intact after cerebellar hemorrhage and monitor the dynamic process of the brainstem intact which changed from being stimulated to being damaged.Purposes1 The stable cerebellar hemorrhage model in rats was established. The characteristics and changing pattern of electronystagmogram spontaneous nystagmus and that being induced by ice water test were summarized and analyzed 72h after model establishment in rats with different degrees of hemorrhage. The pressure of the cisternal magna and spinal subarachnoid space along with their difference were also monitored after the model establishment. It was explored the feasibility of the ENG being applied for monitoring the changing conditions of patients and for anticipating the formation of brain herniation after the cerebellar hemorrhage occurred.2 To observe the neurotransmitter receptors such as GABA-ARα1 and NMDAR1 in brainstem nerve nucleus related eye movements of our rat model, which including paramedian pontine reticular formation (PPRF),vestibular nucleus (VN) and oculor motor nucleus (OMN). To observe the neurochemistry mechanism of the changes of the spontaneous nystagmus and the abnormal nystagmus induced by ice water test through the neurotransmitters receptors of nucleus about eye movements and histological changes being measured in brainstem.3 To explore the pathophysiological mechanisms of the spontaneous nystagmus and the abnormal nystagmus induced by ice water test after cerebellar hemorrhage through oberserving the changes of GABA-ARα1 and NMDAR1 in vestibular nucleus and recording the ENG of the fastigial nucleus (FN) and the flocculonodular lobe (FL) after electrical stimulation and being damaged by electrical coagulation.MethodsPart I: Establishment of the cerebellar hemorrhage model and analysis of ENG1TypeⅦcollagenase was injected into the dentate body of cerebellum in rats under stereotaxis according the method reported by Rosenberg. The rats were randomly divided into five groups:sham group, 0.2U injection group, 0.4U injection group, 0.6U injection group, 0.8U injection group, eight rats in each group.2 The spontaneous nystagmus and nystagmus induced by ice water test were recorded through RM6240C multiple channel electrophysiolograph 72h after the model being established. 3 The pressure of the cisternal magna and spinal subarachnoid space were monitored 72h after the model being established. The pressure difference (dp) between them was also calculated.4 The brain was fixed through 4℃paraformaldehyde perfused into the rats 72h after cerebellar hemorrhage model being established. Cerebellum and brainstem were removed. The coronal section of cerebellum were done by microtome behind and front along the puncture plane. The brainstem coronal sections were made the same as that. The HE staining was performed in the sections for observing pathological change.The hematoma area was calculated through formula.5 Rats were killed 72h after cerebellar hemorrhage model established. The skull was removed and the cerebellum and brainstem were abstracted. Brain water content was measured through WBC way.Part II: The neurochemistrical mechanisms of the spontaneous nystagmus and nystagmus induced by ice water test after cerebellar hemorrhage1 The expressions of NMDAR1 and GABA-ARα1 were observed through immunohistochemistry in PPRF, VN, OMN in brainstem at 72h after cerebellar hemorrhage model being erected.2 The histological and pathological changes of brainstems and cerebellums were observed through HE stainning at 72h after cerebellar hemorrhage model being erected. Part III: spontaneous nystagmus and nystagmus induced by ice water test after the fastigial nucleus and the flocculonodular lobe being electric stimulated and electric coagulated damaged in ratsThe ENG of the spontaneous nystagmus and nystagmus induced by ice water test were recored through RM6240C multiple channel electrophysiolograph of the fastigial nucleus and the flocculonodular lobe after the electrical stimulation and being damaged by electrical coagulation in rats. The expressions of NMDAR1 and GABA-ARα1 were observed through immunohistochemistry in VN.ResultsPart I1 Animal survival rate: 8 rats died among the 40 rats including one for overdose of anesthesia, the others for brain herniation. Animal survival rate was 87.5%.2 The pressure of the cisternal magna and spinal subarachnoid space: The pressure of the cisternal magna and spinal subarachnoid space were different among the 0.2U collagenase group, 0.4U collagenase group, 0.6U collagenase group and 0.8U collagenase group compared with the sham group (P<0.05). The higher doses of collagenase injected, the higher the pressure were, the greater dp between the pressure of cisternal magna and the pressure of spinal subarachnoid space (P<0.05).3 Spontaneous nystagmus 72h after cerebellar hemorrhage in rats: spontaneous nystagmus was not observed in sham group. ENG recorded a smooth sine curve for the eye movements, no sawtooth nystagmus-like. All cerebellar hemorrhage model rats showed spontaneous nystagmus. Rotatory nystagmus were observed in horizontal direction of ENG for the fast phase to the left, slow phase to the right, the vertical upward direction for the fast phase and slow phase-down nystagmus in 0.2U group. 0.4U group also showed rotatory nystagmus, the direction being opposite to that of the 0.2U group. 0.6U group appeared spontaneous nystagmus which the fast phase to left and slow phase to the right, accompanied vertical nystagmus attacking intermittently which the fast phase and slow phase were down or up. The eyes fixed in 0.8U group rats and spontaneous nystagmus were not recorded which ENG showed a straight line.4 Nystagmus induced by ice water 72h after cerebellar hemorrhage in rats: The horizontal nystagmus was induced by one lateral ice water injected to the ear which the fast phase directed to the contralateral to the lateral of ice water being injected and the slow phase directed to the the lateral of ice water being injected in sham group rats. The horizontal nystagmus was induced by ice water injected to the right ear which the fast phase directed to the left and the slow phase directed to the right in 0.2U group. The latency (Lat) was same (P>0.05), the frenquency(F)(P<0.05), the amplitude (Amp), the slow phase velocity (SPV) and the circadian variation index (CVI) were higher compared with sham group (P<0.01). The horizontal nystagmus was induced by ice water injected to the left ear which the fast phase directed to the right and the slow phase directed to the left in 0.2U group. The Lat was longer (P<0.01), F, Amp, SPV were lower (P<0.05), the CVI were higher compared with sham group (P<0.01). The horizontal nystagmus was induced by ice water injected to the right ear which the direction was same to 0.2U group which the Lat was longer (P<0.01), Amp (P<0.05), F, SPV were lower (P<0.01), the CVI were higher compared with sham group (P<0.01) in 0.4U group. While ice water being injected to the left ear, the nystagmus being induced which Lat was same (P>0.05), F, Amp, SPV and the CVI were higher compared with sham group (P<0.01). The horizontal nystagmus was induced by ice water injected to the right ear which the fast phase directed to the left and the slow phase directed to the right which Lat was same (P>0.05), F, Amp (P<0.05), SPV and the CVI were higher compared with sham group (P<0.01) in 0.6U group. The nystagmus was not induced by left ice water test in 0.6U group. Bilateral ice water test were failure in 0.8U group.5 HE staining of the cerebellum in general : The side of the cerebellar hemisphere injected with the collagenase was engorage and full, in which part of them showed downward displacement and the midline shift to the opposite side. Brainstems of part of the rats were distorted. The injection needle track was seen without hematoma and edema in sham group rats. The hematoma was seen in the coronal section of cerebellum of each model rat. The center of hematoma showed madder red and the distinction between the grey and white matter was obscure. The bleeding part was close to each other in the model groups. The greater area of the hematoma was associated with increasing dosage of the collagenase injected (P<0.05).6 Brain water content: The BWC was greater in each model group than the sham group (P<0.05). The correlation between the BWC and the dose of collagenase injection was positive (P<0.05). The injected dosage of collagenase was related to the BWC and cerebral edema.PartⅡ1 Expressions of neurotransmitters in the nucleus about eye movements: The positive cells which membrane was brownish through DAB stained distributed cluster-like in the brainstem. The distribution of inhibitory neurotransmitter ratio (GABA-ARα1/GABA-ARα1+NMDAR1) and excitatory neurotransmitter ratio (NMDAR1/GABA-ARα1+NMDAR1) within PPRF, VN, and OMN of both sides were no difference in the sham group (P>0.05). Excitatory neurotransmitter expressed higher on the right PPRF than the left PPRF in 0.2U collagenase group (P<0.05), which were higher in the left OMN and VN than the right OMN and VN (P<0.05). Excitatory neurotransmitter expressed higher on the left PPRF than the right PPRF in 0.4U collagenase group (P<0.05), which were higher in the right OMN and VN than the left OMN and VN (P<0.05). Excitatory neurotransmitters expressed higher on the right PPRF than the left PPRF in 0.6U collagenase group (P<0.05), excitatory neurotransmitter which were higher in the left OMN and VN than the right OMN and VN (P<0.05). The expressions of the excitory and inhibitory neurotransmitters in the nucleus all decreased in 0.8U group compared with sham and the other model groups. Excitatory neurotransmitters was higher in the right PPRF than in the left (P<0.05), which were higher in the left OMN and VN of than the right (P<0.05).2 The pathological changes of cerebellum and brainstem in rats: The central of the hematoma was seen that the great deal of red cells and inflammatory cells were aggregaed. The neurons were swell which kytoplasm were rarefacted and the structure of the neuro fibries were disorder. The pathological phenomenons were obviously along the more collagenase injected. The HE stainning showed the histology of brainstem were normal in the rats of 0.2U and 0.4U group while were abnormal in the rats which died in 0.6U group and the rats of 0.8U group. The abnomal stainning showed the structure of neuro fibries were discontinued and disordered and the cellular nucleus of neurons were disappeared. The pathological changes showed seriously near the middle line of the brainstem.PartⅢ1 Spontaneous nystagmus and nystagmus induced by ice water after the flocculonodular lobe after electrical stimulation in rats All rats showed spontaneous nystagmus. Rotational nystagmus were observed in horizontal direction of ENG for the fast phase to the left, slow phase to the right, the vertical upward direction for the fast phase and slow phase-down nystagmus. The horizontal nystagmus was induced by ice water injection to the right ear, which the direction and the Lat was no significant difference compared with the control group (P>0.05), whereas the F, Amp, SPV and the CVI were higher compared with the control group (P<0.01). The direction of the nystagmus was opposite to that of the right lateral ice water test by left lateral ice water test, which the Lat was longer (P<0.01), the F, Amp, SPV were lower (P<0.01) and CVI was higher compared with control group (P<0.01).2 Spontaneous nystagmus and nystagmus induced by ice water after the flocculonodular lobe being damaged by electrical coagulation in ratsAll rats showed spontaneous nystagmus. Rotatory nystagmus were observed in horizontal direction of ENG for the fast phase to the right, slow phase to the left, the vertical upward direction for the fast phase and slow phase-down nystagmus. The horizontal nystagmus was induced by ice water injection to the right ear which the direction was same to the control group while the Lat was longer, The F, Amp, SPV were lower compared with control group (P<0.01) and the CVI was higher (P<0.01). The horizontal nystagmus was induced by ice water injected to the left ear which the fast phase directed to the right and the slow phase directed to the left which Lat was same (P>0.05). F, Amp, SPV (P<0.01) and the CVI were higher compared with control group (P<0.01).3 Spontaneous nystagmus and nystagmus induced by ice water after the fastigial nucleus being electric stimulated in ratsAll rats showed spontaneous nystagmus. Rotatory nystagmus were observed in horizontal direction of ENG for the fast phase to the right, slow phase to the left, the vertical upward direction for the fast phase and slow phase-down nystagmus. The horizontal nystagmus was induced by ice water injected to the right ear which the direction was same to the control group which the Lat was longer, the F, Amp, SPV were lower compared with control group (P<0.01) and the CVI was higher (P<0.01). The horizontal nystagmus was induced by ice water injected to the left ear which the fast phase directed to the right and the slow phase directed to the left which Lat was same (P>0.05). The F, Amp, SPV and the CVI were higher compared with control group (P<0.01).4 Spontaneous nystagmus and nystagmus induced by ice water after the fastigial nucleus being damaged by electrical coagulation in ratsAll rats showed spontaneous nystagmus. Rotatory nystagmus were observed in horizontal direction of ENG for the fast phase to the left, slow phase to the right, the vertical upward direction for the fast phase and slow phase-down nystagmus. The horizontal nystagmus was induced by ice water injected to the right ear which the direction and the Lat were no significant difference compared with the control group (P>0.05). The F, Amp, SPV and the CVI were higher compared with the control group (P<0.01). The direction of the nystagmus was opposite to that of the right lateral ice water test by left lateral ice water test, which the Lat was longer, the F, Amp, SPV were lower (P<0.01) and CVI was higher compared with control group (P<0.01).5 After the right flocculonodular lobe being stimulated, excitatory neurotransmitter ratio was lower in the right VN than the left VN (P<0.05). After the destruction of the right flocculonodular lobe, excitatory neurotransmitter ratio was higher in the right VN than the left (P<0.05).6 After the right fastigial nucleus being stimulated, excitatory neurotransmitter ratio was higher in the right VN than the left VN (P <0.05). After the destruction of the right fastigial nucleus, excitatory neurotransmitter ratio was higher in the left VN than the right (P<0.05).Conclusions1 Cerebellar hemorrhage model can be established successfully through the injection of collagenase into cerebellar dentate nuclei of rat under stereotaxis. The hematoma area was increased along the increasing injection dose of collagenase.2 Different forms of spontaneous nystagmus and nystagmus induced by ice water could be showed in the cerebellar hemorrhage model with different amount of bleeding.3 Different amount of bleeding in the cerebellum could affected the brainstem in different extent. Brainstem function could be determined according the different variations of nystagmus after cerebellar hemorrhage.4 PCM, PSSS increased and the pressure difference between them may be the possible causes of changes in nystagmus after cerebellar hemorrhage. |
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