Multiple factors to assist human-derived induced pluripotent stem cells to efficiently differentiate into midbrain dopaminergic neurons

Midbrain dopaminergic neurons play an important role in the etiology of neurodevelopmental and neurodegenerative diseases.They also represent a potential source of transplanted cells for therapeutic applications.In vitro differentiation of functional midbrain dopaminergic neurons provides an accessible platform to study midbrain neuronal dysfunction and can be used to examine obstacles to dopaminergic neuronal development.Emerging evidence and impressive advances in human induced pluripotent stem cells,with tuned neural induction and differentiation protocols,makes the production of induced pluripotent stem cell-derived dopaminergic neurons feasible.Using SB431542 and dorsomorphin dual inhibitor in an induced pluripotent stem cell-derived neural induction protocol,we obtained multiple subtypes of neurons,including 20%tyrosine hydroxylase-positive dopaminergic neurons.To obtain more dopaminergic neurons,we next added sonic hedgehog(SHH)and fibroblast growth factor 8(FGF8)on day 8 of induction.This increased the proportion of dopaminergic neurons,up to 75%tyrosine hydroxylase-positive neurons,with 15%tyrosine hydroxylase and forkhead box protein A2(FOXA2)co-expressing neurons.We further optimized the induction protocol by applying the small molecule inhibitor,CHIR99021(CHIR).This helped facilitate the generation of midbrain dopaminergic neurons,and we obtained 31-74%midbrain dopaminergic neurons based on tyrosine hydroxylase and FOXA2 staining.Thus,we have established three induction protocols for dopaminergic neurons.Based on tyrosine hydroxylase and FOXA2 immunostaining analysis,the CHIR,SHH,and FGF8 combined protocol produces a much higher proportion of midbrain dopaminergic neurons,which could be an ideal resource for tackling midbrain-related diseases.

In vitro culture and differentiation of rat embryonic midbrain-derived neural stem cells

BACKGROUND： Midbrain-derived neural stem cells （mNSCs） can differentiate into functional mature dopaminergic neurons. The mNSCs are considered the ideal choice for cell therapy of Parkinson＇s disease. OBJECTIVE： To isolate rat embryonic mNSCs and to observe the differentiation characteristics of mNSCs induced by cell growth-promoting factors. DESIGN, TIME AND SETTING： An in vitro cell culture study based on the molecular biology of nerve cells was carried out at the Institute of Clinical Medicine, China-Japan Friendship Hospital （China） from March to November 2007. MATERIALS： Sprague Dawley rats at embryonic day 14 were used in this study. Nestin antibody, β-Ⅲ tubulin antibody, glial fibrillary acidic protein （GFAP） antibody and cyclic nucleotide 3＇-phosphohydrolase （CNPase） antibody were provided by Abcam; DMEM/F12 medium and N2 supplement were provided by Invitrogen; epidermal growth factor （EGF） and fibroblast growth factor-2 （FGF2） were provided by R＆D Systems. METHODS： The ventral mesencephalon was dissected from embryonic day 14 rat embryos. By trypsin digestion and mechanical separation, the brain tissue was triturated into a fine single-cell suspension. The cells were cultured in 5 mL serum-free medium containing DMEM/FI 2, 1% N： supplement, 20 ng/mL EGF and FGF2. The mNSCs at the third generation were coated with 10ug/mL polylysine and induced to differentiate in the DMEM/F12 supplemented with 1% fetal bovine serum and 1% N2. MAIN OUTCOME MEASURES： The neural spheres of the third passage were identified by nestin immunofluorescence; at the same time, the cells were induced to differentiate, and the types of differentiated cell were identified by immunofluorescence for β Ⅲ tubulin, GFAP and CNPase. RESULTS： Seven days after primary culture, a great many neurospheres could be obtained by successive pasage. Immunofluorescence assays showed that the neurospheres were nestin positive, and after differentiation, the cells expressed GFAP, CNPase and β -Ⅲ-tubulin. CONCLUSION： Embryonic day 14 rat mNSCs can differentiate into neuron-like cells and glial cells following induction by EGF, FGF2 and N： additive.

Magnesium effects on behavior and substance P mRNA expression in the midbrain of a rat migraine model

BACKGROUND： Substance P participates in pain transmission and modulation, suggesting a close association with migraine headaches. The clinical application of magnesium has been effective in treating migraines, and the action mechanisms underlying migraines correlate with substance P expression. OBJECTIVE： To analyze different magnesium doses on behavior and substance P mRNA expression in the midbrain of a rat migraine model, and to determine the action pathway of migraine treatment using magnesium. DESIGN, TIME AND SETTING： A completely randomized, controlled, animal experiment was performed at the Experimental Animal Center and Central Laboratory in the Second Hospital of Jilin University between 2007 and 2008. MATERIALS： Magnesium sulfate （25%） was supplied by Tianjin Pharmaceutical Jiaozuo, China. Nitroglycerin was provided by Shanxi Kangbao Biological, China. Substance P primer sequence was synthesized by TaKaRa Biotechnology （Dalian）, China. METHODS： A total of 36 healthy, adult, Wistar rats were randomly assigned to 6 groups： control, migraine, low- and high-dose magnesium sulfate treated, and low- and high-dose magnesium sulfate control, with 6 rats in each group. Migraines were induced by subcutaneous injection of 10 mg/kg nitroglycerin in the migraine and low- and high-dose magnesium sulfate treated groups, and 2 mL/kg physiological saline was administered to rats in the control and low- and high-dose of magnesium sulfate control groups. Five minutes following administration, rats in low-dose groups were intraperitoneally injected with 100 mg/kg magnesium sulfate, while those in high-dose groups were injected with 300 mg/kg magnesium sulfate. No interventions were administered to the control and migraine groups. MAIN OUTCOME MEASURES： At 2 hours after nitroglycerin injection, substance P mRNA expression in the rat midbrain was measured by real-time quantitative polymerase chain reaction. At 60-90 minutes after nitroglycerin injection, behavioral changes of pain were analyzed in the experimental rats. RESULTS： The migraine group exhibited significantly lower levels of substance P mRNA expression compared with the control group （P 〈 0.05）. Following magnesium sulfate injection, substance P mRNA expression increased, compared with the migraine and control groups （P 〈 0.05）. In the low- and high-dose magnesium sulfate treated groups, pain behavior was remarkably ameliorated, compared with the migraine group （P 〈 0.05）, particularly with the high-dose injection （P 〈 0.05）. CONCLUSION： Magnesium relieved pain behaviors in a rat migraine model in a dose-dependent manner, and the therapeutic effect was achieved in conjunction with increased substance P expression in the midbrain.

The neuroprotective and regenerative potential of parkin and GDNF/Ret signaling in the midbrain dopaminergic system

Parkinson＇s disease（PD）is the second most common neurodegenerative disease after Alzheimer＇s disease.The etiology of PD is still not completely understood,but the degeneration of dopaminergic（DA）neurons in the substantia nigra pars compacta（SNpc）,loss of DA innervation of the striatum,and protein aggregates in the form of Lewy bodies and neurites are its established hallmarks. In addition to α-synuclein accumu- lation in Lewy bodies and neurites, genetic mutations in the genes encoding parkin, PINK, DJ-1, LRRK2 and other proteins are associated with the inherited form of PD. An association study linked also the receptor tyrosine kinase Ret to PD （Meka et al., 2015）. Currently there are only symptomatic treatments available for PD but no cure. Consequently much effort is being made to find neurotrophic and other factors able to stimulate SNpc DA neuron protection and regeneration.

Special type of Wernekink syndrome in midbrain infarction:Four case reports

BACKGROUND Wernekink commissural syndrome(WCS)is a distinct midbrain syndrome that involves the caudal tegmentum of the midbrain and selectively damages the Wernekink commissure involved in the decussation of the superior cerebellar peduncle in midbrain.The aim of the study was to explore the clinical manifestations,imaging characteristics,and differential diagnosis of WCS in midbrain infarction to provide reference for clinicians in the diagnosis of WCS.CASE SUMMARY The clinical data of 4 patients with WCS with midbrain infarction were analyzed retrospectively.WCS is a rare syndrome that can be diagnosed based on its characteristic symptoms and imaging findings of magnetic resonance imaging.CONCLUSION Clinicians should look for this syndrome in cases of bilateral cerebellar dysfunction and eye movement disorders.

Tyrosine hydroxylase expression in the midbrain of Parkinson's disease model rats treated with Xifeng Dingchan decoction

This study showed that abnormal behavioral changes were greatly improved in rats displaying Parkinson＇s disease-like symptoms after intragastric administration of Xifeng Dingchan decoction at 15, 7.5, 3.75 g/kg per day. In addition, tyrosine hydroxylase mRNA expression in the substantia nigra of the midbrain was up-regulated, and tyrosine hydroxylase content in the midbrain ventral tegmentum and substantia nigra pars compacta was also increased. The effect of administration of Xifeng Dingchan decoction at 7.5 g/kg per day was similar to that of Madopar at 67.5 mg/kg per day. These results indicate that the therapeutic effect of Xifeng Dingchan decoction on Parkinson＇s disease is associated with the up-regulated protein and mRNA expression of tyrosine hydroxylase in the midbrain.

Dorsal midbrain syndrome secondary to pineal gland tumours: case series and review on manifestations, management and outcome

Dorsal midbrain syndrome or Parinaud syndrome is a supranuclear brainstem syndrome involving the vertical gaze centre.These are case series with three patients who were diagnosed with dorsal midbrain syndrome secondary to pineal gland tumours.The prognosis varied depending on tumour types,age of presentation and treatment received.All of them were presented with life-threatening obstructive hydrocephalus.Our first patient was successfully treated with emergency surgery followed by radiotherapy.He regained normal visual acuity and full recovery of his ocular movement.Second and third patients had undergone surgery for raised intracranial pressure.Both had an inoperable pineal gland tumour.As for our second patient,we detected a worsening of vertical gaze during his four years follow-up.However,his bilateral good visual acuity was preserved.The third patient passed away as a result of uncontrolled enlarging tumour.We also briefly reviewed the clinical presentation,diagnosis,and therapeutic approach of the three patients.One of the caveats is that urgent radiological study is crucial to differentiate the tumour type via the pathognomonic features and to delineate the tumour extension.The preferable treatment options vary among each tumour type.A multidisciplinary approach is crucial in early detection,in addition to treatment initiation and long term follow up to achieve a better outcome.

Targeting transcriptional regulators to regenerate midbrain dopaminergic axons in Parkinson's disease

Introduction： Parkinson＇s disease （PD） is a chronic, age-re- lated neurodegenerative disorder that affects 1-2% of the population over the age of 65. PD is characterised by the progressive degeneration of nigrostriatal dopaminergic （DA） neurons. This leads to disabling motor symptoms, due to the striatal DA denervation. Despite decades of research, there is still no therapy that can slow, stop or regenerate the dying midbrain DA neurons in PD.

Prenatal and Postnatal Exposures to 1-Methyl-4-phenyl-1,2,3,6-tetra Hydropyridine (MPTP) Impaired Mouse Midbrain Dopamine System and May Produce a Predisposing and Inducing Model for Parkinson’s Disease

Dopamine cell bodies in the substantia nigra of the midbrain and with their terminals projecting to the neostriatum form the nigrostriatum and these dopamine neurons degenerate in Parkinson’s disease (PD). Based on metabolic and func- tional specialization of the cell bodies versus the axon terminals, the level and disposition of dopamine, its metabolites and enzymes are different in both regions and are likely to be affected differently in PD. We examined changes in the midbrain dopamine system following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), to test the hypothesis that a predisposing/sensitization stage and a inducing/precipitating stage underlie PD. Pregnant mice were treated with a low dose of MPTP during gestation days 8 - 12 to model the predisposing/sensitization stage, by interrupting the fetal mid- brain dopamine system during its neurogenesis. For the inducing/precipitating stage, the 12-weeks offspring were ad- ministered MPTP. The prenatal-MPTP offspring appear normal, but midbrain dopamine, 3,4-di-hydroxy-phenyl-acetic- acid, 3-methoxytyramine, tyrosine-hydroxylase and L-aromatic-amino-acid-decarboxylase, were reduced by 49.6%, 48%, 54%, 20.9% and 25%. Postnatal-MPTP of 10, 20, 30 mg/kg administered to the prenatal-PBS vs prenatal-MPTP offspring reduced midbrain dopamine by 43.6%, 47.2%, 70.3% vs 85.4%, 89.1%, 95.2%;tyrosine-hydroxylase by 30%, 63%, 81% vs 30.7%, 70.4%, 91.4%;L-aromatic-amino-acid-decarboxylase by 0%, 2%, 40% vs 32%, 40%, 58%. The prenatal-MPTP may render the DA system sensitive by causing sub-threshold reduction of DA, its metabolites and en- zymes, enabling postnatal-MPTP to reduce dopamine above the 70% - 80% PD-inducing threshold. Thus, the study may produce a prenatal predisposing/sensitization and postnatal inducing/precipitation model of PD. It also indicates that some cases of PD may have a fetal basis, in which sub-threshold nigrostriatal impairments occur early in life and PD-symptoms are induced during aging by further insults to the dopaminergic system that would not cause PD symptoms in normal indi-viduals.

A Rare Case of Isolated Left Medial Midbrain Stroke

Midbrain stroke is uncommon. We are presenting an interesting case of a patient with rare isolated left midbrain stroke. The patient had third cranial nerve palsy. Magnetic resonance imaging (MRI) brain showed acute stroke at the medial part of left midbrain. Magnetic resonance angiography (MRA) was normal.

Transforming growth factor β1-mediated anti-inflammation slows progression of midbrain dopaminergic neurodegeneration in Parkinson's disease?

Parkinson’s disease（PD）is characterized by the progressive loss of midbrain dopaminergic（m DA）neurons and a subsequent decrease in striatal dopamine levels,which cause the typical clinical motor symptoms such as muscle rigidity,bradykinesia and tremor.

Holmes tremor in the head and neck region caused by midbrain and thalamic hemorrhage:a case report

Objective:to describe the clinical fea-tures of Holmes tremor(HT)in the head and neck region caused by midbrain and thalamus hemorrhage.Methods:we collected the clinical history and examination data of a patient with HT in the head and neck region.Results:the patient had HT in the head and neck region with dizzi-ness.Brain computed to mography(CT)showed midbrain and thalamus hemorrhage.Conclusion:HT in the head and neck region as a delayed complication of midbrain and thalamus stroke are rare in clinic.When patients have a tremor in the head and neck region after midbrain or thalamus stroke,HT should be considered.

抑制dmPAG区谷氨酸能神经元可减轻创伤后应激障碍小鼠的过度防御反应

目的探索背内侧中脑导水管周围灰质(dmPAG)谷氨酸能神经元对创伤后应激障碍(PTSD)小鼠过度防御行为的调控作用。方法84只雄性C57BL/6J小鼠随机分为12组(7只/组)。研究PTSD后过度防御反应时,分为CON组和PTSD组;研究激活dmPAG区谷氨酸能神经元对PTSD后过度防御反应的影响,分为CON NS组,CON mCherry组,PTSD mCherry组和PTSD hM3Dq组;研究抑制dmPAG区谷氨酸能神经元对PTSD后过度防御反应的影响,分为CON NS组,CON mCherry组,PTSD mCherry组和PTSD hM4Di组。构建单效长时程应激模型(SPS)模拟PTSD状态,通过视觉本能恐惧实验、触须刺激实验、条件恐惧实验测试小鼠的防御行为,免疫荧光染色观察dmPAG区谷氨酸能神经元活性变化。结果与CON组相比,PTSD组回巢潜伏期缩短(P<0.001),巢内停留时间百分比增加(P<0.01),防御评分增加(P<0.001),僵直时间百分比增加(P<0.01)。免疫荧光染色结果可见PTSD小鼠防御行为过程中,dmPAG区c-fos阳性的谷氨酸能神经元占比明显增加(P<0.001)。与PTSD mCherry组相比,激活dmPAG区谷氨酸能神经元后,PTSD hM3Dq组回巢潜伏期、巢内停留时间百分比无差异,防御评分升高(P<0.05),僵直时间百分数增加(P<0.01)。与PTSD mCherry组相比,抑制dmPAG区谷氨酸能神经元后,PTSD hM4Di组回巢潜伏期延长(P<0.05),巢内停留时间百分比降低(P<0.05),防御评分降低(P<0.01),僵直时间百分比降低(P<0.01)。结论抑制dmPAG区谷氨酸能神经元活性可以明显减轻PTSD的过度防御行为。

PERK通路在内质网应激诱导的中脑多巴胺能神经元死亡中作用

目的 探讨PERK通路在内质网应激诱导的中脑多巴胺能神经元变性死亡中的作用。方法 利用小鼠原代培养成纤维细胞,应用Western blot和免疫荧光染色法,观察内质网应激诱导剂毒胡萝卜素(thapsigargin)对PERK通路的激活,利用小鼠原代培养中脑多巴胺能神经元,应用TH免疫荧光染色法对存活的中脑多巴胺能神经元进行计数,以观察PERK抑制剂GSK2606414对长时程Thapsigargin处理诱导的中脑多巴胺能神经元死亡的影响。结果 在Thapsigargin处理后的原代成纤维细胞中p-PERK、p-eIF2α、ATF4水平均显著升高,Thapsigargin诱导中脑多巴胺能神经元死亡,PERK抑制剂GSK2606414可显著减少长时程Thapsigargin处理诱导的中脑多巴胺能神经元死亡。结论 内质网应激激活PERK通路;长时程内质网应激导致中脑多巴胺能神经元死亡,抑制PERK通路的激活可以缓解长时程内质网应激导致的中脑多巴胺能神经元死亡,PERK通路参与了长时程内质网应激导致的中脑多巴胺能神经元变性死亡。

Holmes' tremor caused by midbrain cavernoma

Holmes＇ tremor has been postulated as a syndrome .attributed to those lesions that interrupt the dentatethalamic and the nigrostriatal tracts thus causing both an action and a rest tremor. It may arise from various underlying structural disorders including multiple sclerosis, stroke, or tumors. So far, to our knowledge, few studies on Holmes＇ tremor secondary to cavemoma have been reported. Here we report a case of disabling tremor, who harbored a cavemoma in the midbrain.

A Novel Movement Behavior Control Method for Carp Robot through the Stimulation of Medial Longitudinal Fasciculus Nucleus of Midbrain

Biological robot is a kind of creature controlled by human beings by applying intervention signals through control technology to regulate biological behavior.At present,the research on bio-robot mainly focuses on terrestrial mammals and insects,while the research on aquatic animal robot is less.Early studies have shown that the medial longitudinal fasciculus nucleus(NFLM)of carp midbrain was related to tail wagging,but the research has not been applied to the navigation control of the carp robot.The purpose of this study is to realize the quantitative control of the forward and steering behavior of the carp robot by NFLM electrical stimulation.Under the condition of no craniotomy,brain electrode was implanted into the NFLM of the carp midbrain,and the underwater control experiment was carried out by applying different electrical stimulation parameters.Using the ImageJ software and self-programmed,the forward motion speed and steering angle of steering motion of the carp robot before and after being stimulated were calculated.The experimental results showed for the carp robot that was induced the steering motion,the left and right steering motion of 30°to 150°could be achieved by adjusting the stimulation parameters,for the carp robot that was induced the forward motion,the speed of forward motion could be controlled to reach 100 cm/s.The research lays a foundation for the accurate control of the forward and steering motion of the aquatic animal robot.

Central processing of itch in the midbrain reward center

With the support by the National Natural Science Foundation of China,the research team jointly directed by Prof.Xu TianLe(徐天乐)at the Department of Anatomy and Physiology,Shanghai Jiao Tong University School of Medicine,and Prof.Hu Ji(胡霁)at the School of Life Science and Technology,ShanghaiTech University,recently reported in Neuron as a research article the midbrain circuit mechanisms underlying different components of the itch-scratch cycles(Neuron,2019,102:1—15).

佩里综合征动物模型多巴胺转运体缺陷的超高分辨率显微成像研究

目的探讨佩里综合征模型小鼠中脑多巴胺能神经元纹状体多巴胺转运体(DAT)缺陷的亚细胞机制。方法利用免疫荧光染色法,采用Airyscan超高分辨率显微成像和三维成像技术,检测佩里综合征模型小鼠中脑多巴胺能神经元中DAT表达分布情况。结果佩里综合征模型小鼠中脑多巴胺能神经元部分树突发生病理性肿胀,而在这些肿胀中有荧光强度显著增加的异常内质网结构,以及异常增多的溶酶体。大量的DAT呈点状定位于树突异常肿胀内,且有相当数量的DAT与异常内质网结构,或异常增多的溶酶体共定位。结论佩里综合征模型小鼠中脑多巴胺能神经元中DAT潴留于树突肿胀内质网中,不能有效的输出和运送至纹状体发挥功能;同时树突肿胀中DAT降解增多,进一步加剧了纹状体DAT缺陷。

中脑梗死导致Wernekink连合综合征1例报告

脑梗死是最常见的缺血性脑卒中类型,临床中孤立性的中脑梗死相对少见,而Wernekink连合综合征是一种特殊类型的中脑梗死,主要表现为双侧小脑性共济失调及眼肌麻痹,该病临床罕见,现将我科收治的1例类似Miller-Fisher综合征表现的中脑梗死所致Wernekink连合综合征报道如下。1临床资料患者,男,48岁。因“视物重影4 d,肢体活动不灵活、言语不清1 d”于2022年6月6日入院。患者于入院4 d前早上7点起床后感到视物重影,双眼视物时有水平方向重影,单眼视物清晰。患者未予重视,上述症状持续不缓解,1 d前开始出现言语不清、语调低沉,肢体活动不灵活,表现为肢体动作不协调,如夹菜、刷牙等动作不准确,同时伴有步态不稳,漂浮感。无头晕、头痛,无肢体麻木、乏力等。就诊于广州医科大学附属第六医院急诊科,急诊头部CT未见明显异常,以“复视查因”收入神经内科病房。