AAV-Mediated Astrocyte-Specific Gene Expression under Human ALDH1L1 Promoter in Mouse Thalamus

Adeno-associated virus (AAV)-mediated gene delivery has been proposed to be an essential tool of gene therapy for various brain diseases. Among several cell types in the brain, astrocyte has become a promising therapeutic target for brain diseases, as more and more contribution of astrocytes in pathophysiology has been revealed. Until now, genetically targeting astrocytes has been possible by utilizing the glial fibrillary acidic protein (GFAP) promoter. In some brain areas including thalamus, however, the GFAP expression in astrocytes is reported to be low, making it difficult to genetically target astrocytes using GFAP promoter. To study the function of astrocytes in thalamus, which serves as a relay station, there is a great need for identifying an alternative astrocyte-specific promoter in thalamus. Recently, a new astrocyte-specific promoter of ALDH1L1 has been identified. However, it has not been examined in thalamus. Here we developed and characterized an AAV vector expressing Cre recombinase under the human ALDH1L1 promoter, AAV-hALDH1L1-Cre. To test the cell-type specific expression of AAV-hALDH1L1-Cre, AAV virus was injected into several brain regions of Ai14 (RCL-tdTomato) mouse, which reports Cre activity by tdTomato expression. In thalamus, we observed that tdTomato was found mostly in astrocytes (91.71%), with minimal occurrence in neurons (2.67%). In contrast, tdTomato signal was observed in both neurons and astrocytes of the amygdala (neuron: 68.13%, astrocyte: 28.35%) and hippocampus (neuron: 76.25%, astrocyte: 18.00%), which is consistent with the previous report showing neuronal gene expression under rat ALDH1L1 promoter. Unexpectedly, tdTomato was found mostly in neurons (91.98%) with minimal occurrence in astrocytes (6.66%) of the medial prefrontal cortex. In conclusion, hALDH1L1 promoter shows astrocyte-specificity in thalamus and may prove to be useful for targeting thalamic astrocytes in mouse.

Differential modulation of electrical stimulation of periaqueductal gray and thalamus on nociceptive behaviors of rats / 生理学报

Deep brain stimulation (DBS) is a surgical treatment which has shown remarkable therapeutic benefits for patients with a variety of neurologic conditions. As an important application, DBS has been used to treat intractable pain for over 60 years. Clinical studies have revealed that the selection of the stimulation sites depended on the types of pain. In this study, we selected ventrolateral periaqueductal gray (vlPAG) and ventral posterior lateral nucleus (VPL) as the target brain areas, which were widely used in clinical treatment of refractory pain, to clarify and compare the effects of vlPAG and VPL stimulation on different models of pain. Acute pain was evoked by thermal stimulation. The chronic inflammatory pain was produced by complete Freund's adjuvant (CFA) injection, while neuropathic pain was induced by spinal nerve ligation (SNL) surgery. Some important results emerged from this study: (1) in the experiment of normal rats, we found that unilateral vlPAG stimulation could lead to a significant increase of the thermal withdrawal threshold in bilateral hindpaws of rats, which means a significant bilateral analgesic action; (2) in the CFA test, both contralateral vlPAG and VPL stimulation significantly alleviated the thermal hyperalgesia, which exhibited analgesic effects to the chronic inflammatory pain; (3) in the SNL experiment, the results revealed that contralateral VPL stimulation could significantly abolish the mechanical allodynia induced by SNL, indicating remarkable analgesic effect to neuropathic pain. But the vlPAG stimulation did not have any effect on the mechanical allodynia. These results suggest that the electrical stimulation of the PAG works more effectively on nociceptive pain, including acute pain and chronic inflammatory pain. Besides, the VPL stimulation is much more sensitive for chronic pain, including chronic inflammatory pain and neuropathic pain.

The Neuromodulation of Neuropathic Pain by Measuring Pain Response Rate and Pain Response Duration in Animal

OBJECTIVE: Neuropathic pain causes patients feel indescribable pain. Deep Brain Stimulation (DBS) is one of the treatment methods in neuropathic pain but the action mechanism is still unclear. To study the effect and mechanism of analgesic effects from DBS in neuropathic pain and to enhance the analgesic effect of DBS, we stimulated the ventral posterolateral nucleus (VPL) in rats. METHODS: To observe the effect from VPL stimulation, we established 3 groups : normal group (Normal group), neuropathic pain group (Pain group) and neuropathic pain+DBS group (DBS group). Rats in DBS group subjected to electrical stimulation and the target is VPL. RESULTS: We observed the behavioral changes by DBS in VPL (VPL-DBS) on neuropathic pain rats. In our study, the pain score which is by conventional test method was effectively decreased. In specific, the time of showing withdrawal response from painful stimulation which is not used measuring method in our animal model was also decreased by DBS. CONCLUSION: The VPL is an effective target on pain modulation. Specifically we could demonstrate changes of pain response duration which is not used, and it was also significantly meaningful. We thought that this study would be helpful in understanding the relation between VPL-DBS and neuropathic pain.

Ginsenosides Have a Suppressive Effect on c-Fos Expression in Brain and Reduce Cardiovascular Responses Increased by Noxious Stimulation to the Rat Tooth

The purpose of this study is to investigate the antinociceptive effects of ginsenosides on toothache. c-Fos immunoreactive (IR) neurons were examined after noxious intrapulpal stimulation (NS) by intrapulpal injection of 2 M KCl into upper and lower incisor pulps exposed by bone cutter in Sprague Dawley rats. The number of Fos-IR neurons was increased in the trigeminal subnucleus caudalis (Vc) and the transitional region between Vc and subnucleus interpolaris (Vi) by NS to tooth. The intradental NS raised arterial blood pressure (BP) and heart rate (HR). The number of Fos-IR neurons was also enhanced in thalamic ventral posteromedial nucleus (VPMN) and centrolateral nucleus (CLN) by NS to tooth. The intradental NS increased the number of Fos-IR neurons in the nucleus tractus solitarius (NTS) and rostral ventrolateral medulla (RVLM), hypothalamic supraoptic nucleus (SON) and paraventricular nucleus (PVN), central cardiovascular regulation centers. Ginsenosides reduced the number of c-Fos-IR increased by NS to tooth in the trigeminal Vc and thalamic VPMN and CLN. Naloxone, an opioid antagonist, did not block the effect of ginsenoside on the number of Fos-IR neurons enhanced by NS to tooth in the trigeminal Vc and thalamic VPMN and CLN. Ginsenosides ameliorated arterial BP and HR raised by NS to tooth and reduced the number of Fos-IR neurons increased by NS to tooth in the NTS, RVLM, hypothalamic SON, and PVN. These results suggest that ginsenosides have an antinociceptive effect on toothache through non-opioid system and attenuates BP and HR increased by NS to tooth.

Sonographic assessment of vessel invasion by cervical masses in dogs / Avaliação ultrassonográfica da invasão vascular por massas cervicais em cães

Tumor invasion of the vessels displays both therapeutic and prognostic implications and represents a challenge for head and neck surgeons. Although previous research has shown that ultrasound can detect such invasions, accurate sonographic parameters to do so have not yet been established. We sought to determine sonographic criteria which are able to characterize these invasions. A high-resolution transducer was used to perform ultrasound examinations of 15 patients selected from a group with inconclusive radiography and computed tomography diagnosis. We found that encasement of the vessel, tumor immobility or fixation in the vessel wall, and narrowing and/or deformity of the lumen were the best criteria. Indeed, when loss of hyperechoic interface of the vessel wall was used as a single criterion it generated false positive results. This study shows that a combination of parameters can be used to provide the best sensitivity and specificity values to produce conclusive diagnosis of vessel invasion by tumors in the cervical region.

Determinaram-se critérios ultrassonográficos capazes de caracterizar a invasão vascular por tumores em cães. Utilizaram-se transdutores de alta resolução para os exames ultrassonográficos realizados em 15 pacientes, selecionados de um grupo submetido previamente à radiografia e tomografia computadorizada, com resultados inconclusivos. Os melhores critérios encontrados foram: encarceramento do vaso, imobilidade do tumor ou aderência na parede vascular e estreitamento ou deformidade luminal. A perda de definição da interface hiperecoica da parede vascular quando foi usada como critério isolado produziu resultados falso positivos. O estudo demonstrou que uma combinação de parâmetros pode ser usada para aumentar a sensibilidade e especificidade diagnóstica, produzindo diagnósticos mais conclusivos e precisos pra definir a invasão vascular por tumores na região cervical ventral.

Effects and mechanism of low frequency stimulation of pedunculopontine nucleus on spontaneous discharges of ventrolateral thalamic nucleus in rats / 生理学报

Parkinson's disease is a progressive neurodegenerative disorder characterized clinically by rigidity, akinesia, resting tremor and postural instability. It has recently been suggested that low frequency stimulation of the pedunculopontine nucleus (PPN) has a role in the therapy for Parkinsonism, particularly in gait disorder and postural instability. However, there is limited information about the mechanism of low frequency stimulation of the PPN on Parkinson's disease. The present study was to investigate the effect and mechanism of low frequency stimulation of the PPN on the firing rate of the ventrolateral thalamic nucleus (VL) in a rat model with unilateral 6-hydroxydopamine lesioning of the substantia nigra pars compacta. In vivo extracellular recording and microiontophoresis were adopted. The results showed that the firing rate of 60.71% VL neurons in normal rats and 59.57% VL neurons in 6-hydroxydopamine lesioned rats increased with low frequency stimulation of the PPN. Using microiontophoresis to VL neurons, we found the firing rate in VL neurons responded with either an increase or decrease in application of acetylcholine (ACh) in normal rats, whereas with a predominant decrease in M receptor antagonist atropine. Furthermore, the VL neurons were mainly inhibited by application of γ-aminobutyric acid (GABA) and excited by GABA(A) receptor antagonist bicuculline. Importantly, the VL neurons responding to ACh were also inhibited by application of GABA. We also found that the excitatory response of the VL neurons to the low frequency stimulation of the PPN was significantly reversed by microiontophoresis of atropine. These results demonstrate that cholinergic and GABAergic afferent nerve fibers may converge on the same VL neurons and they are involved in the effects of low frequency stimulation of the PPN, with ACh combining M(2) receptors on the presynaptic membrane of GABAergic afferents, which will inhibit the release of GABA in the VL and then improve the symptoms of Parkinson's disease.

Neuronal firing in the ventrolateral thalamus of patients with Parkinson's disease differs from that with essential tremor / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Although thalamotomy could dramatically improve both parkinsonian resting tremor and essential tremor (ET), the mechanisms are obviously different. This study aimed to investigate the neuronal activities in the ventrolateral thalamus of Parkinson's disease (PD) and ET.</p><p><b>METHODS</b>Thirty-six patients (PD: 20, ET: 16) were studied. Microelectrode recordings in the ventral oral posterior (Vop) and the ventral intermediate nucleus (Vim) of thalamus was performed on these patients who underwent thalamotomy. Electromyography (EMG) was recorded simultaneously on the contralateral limbs to surgery. Single unit analysis and the interspike intervals (ISIs) were measured for each neuronal type. ISI histogram and auto-correlograms were constructed to estimate the pattern of neuronal firing. Mann-Whitney test and Kruskal-Wallis (K-W) test were used to compare the mean spontaneous firing rate (MSFR) of neurons of PD and ET patients.</p><p><b>RESULTS</b>Three hundred and twenty-three neurons were obtained from 20 PD trajectories, including 151 (46.7%) tremor related neuronal activity, 74 neurons (22.9%) with tonic firing, and 98 (30.4%) neurons with irregular discharge. One hundred and eighty-seven neurons were identified from 16 ET trajectories including 46 (24.6%) tremor-related neuronal activity, 77 (41.2%) neurons with tonic firing, and 64 neurons (34.2%) with irregular discharge. The analysis of MSFR of neurons with tonic firing was 26.7 (3.4 - 68.3) Hz (n = 74) and that of neurons with irregular discharge (n = 98) was 13.9 (3.0 - 58.1) Hz in PD; whereas MSFR of neurons with tonic firing (n = 77) was 48.8 (19.0 - 135.5) Hz and that of neurons with irregular discharge (n = 64) was 26.3 (8.7 - 84.7) Hz in ET. There were significant differences in the MSFR of two types of neuron for PD and ET (K-W test, both P < 0.05). Significant differences in the MSFR of neuron were also obtained from Vop and Vim of PD and ET (16.3 Hz vs. 34.8 Hz, 28.0 Hz vs. 49.9 Hz) (K-W test, both P < 0.05), respectively.</p><p><b>CONCLUSION</b>In consistent with recent findings, the decreased MSFR of neurons observed in the Vop is likely to be involved in PD whereas the increased MSFR of neurons seen in the Vim may be a cause of ET.</p>

Neuronal firing in the globus pallidus internus and the ventrolateral thalamus related to parkinsonian motor symptoms / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>It has been proposed that parkinsonian motor signs result from hyperactivity in the output nucleus of the basal ganglia, which suppress the motor thalamus and cortical areas. This study aimed to explore the neuronal activity in the globus pallidus internus (GPi) and the ventrolateral thalamic nuclear group (ventral oral posterior/ventral intermediate, Vop/Vim) in patients with Parkinson's disease (PD).</p><p><b>METHODS</b>Twenty patients with PD who underwent neurosurgery were studied. Microelectrode recording was performed in the GPi (n = 10) and the Vop/Vim (n = 10) intraoperatively. Electromyography (EMG) contralateral to the surgery was simultaneously performed. Single unit analysis was carried out. The interspike intervals (ISI) and coefficient of variation (CV) of ISI were calculated. Histograms of ISI were constructed. A unified Parkinson's disease rating scale (UPDRS) was used to assess the clinical outcome of surgery.</p><p><b>RESULTS</b>Three hundred and sixty-three neurons were obtained from 20 trajectories. Of 175 GPi neurons, there were 15.4% with tremor frequency, 69.2% with tonic firing, and 15.4% with irregular discharge. Of 188 thalamic neurons, there were 46.8% with tremor frequency, 22.9% with tonic firing, and 30.3% with irregular discharge. The numbers of three patterns of neuron in GPi and Vop/Vim were significantly different (P < 0.001). ISI analysis revealed that mean firing rate of the three patterns of GPi neurons was (80.9 +/- 63.9) Hz (n = 78), which was higher than similar neurons with 62.9 Hz in a normal primate. For the Vop/Vim group, ISI revealed that mean firing rate of the three patterns of neurons (n = 95) was (23.2 +/- 17.1) Hz which was lower than similar neurons with 30 Hz in the motor thalamus of normal primates. UPDRS indicated that the clinical outcome of pallidotomy was (64.3 +/- 29.5)%, (83.4 +/- 19.1)% and (63.4 +/- 36.3)%, and clinical outcome of thalamotomy was (92.2 +/- 12.9)%, (68.0 +/- 25.2)% and (44.3 +/- 27.2)% for tremor, rigidity and bradykinesia, respectively. A significant difference of tremor and rigidity was found between GPi and Vop/Vim (P < 0.05).</p><p><b>CONCLUSIONS</b>Different changes in neuronal firing rate and the pattern in GPi and Vop/Vim are likely responsible for parkinsonian motor signs. The results support the view that abnormal neuronal activity in GPi and Vop/Vim are involved in the pathophysiology of parkinsonism.</p>

Effects of unilateral lesion of the nigrostriatal pathway by 6-OHDA on the neuronal activities of the pedunculopontine nucleus and the ventrolateral thalamic nucleus / 中国应用生理学杂志

<p><b>AIM</b>To investigate the changes in neuronal activities of the pedunculopontine nucleus (PPN) and the ventrolateral thalamic nucleus (VL) after unilateral 6-hydroxydopamin (6-OHDA) lesioning of the striatum in rats.</p><p><b>METHODS</b>Extracellular single-unit recordings were perin normal rats and 6-OHDA lesioned rats to observe the firing rate and firing pattern occurring in PPN and VL neurons.</p><p><b>RESULTS</b>The firing rate of PPN neurones significantly increased from (8.31 +/- 0.62) Hz in normal rats to (10.70 +/- 0.85) Hz in 6-OHDA lesioned rats. The firing pattern changed towards more irregular and bursty when compared with the normal rats, with the firing rate increasing in regular pattern. The firing rate of VL neurones in normal rats and 6-OHDA lesioned rats were (6.25 +/- 0.54) Hz and (5.67 +/- 0.46)Hz respectively, whereas to normal animals. Surthere were no significant differences in these two groups. In addition, the firing pattern did not change in VL compared prisingly, the firing rate in burst pattern decreased significantly.</p><p><b>CONCLUSION</b>These findings demonstrate that PPN neurons are overactive in 6-OHDAlesioned rats, indicating the participation of this nucleus in the pathophysiology of parkinsonism and the activities of VL neurons might be regulated by projection from PPN to VL.</p>

Effects of electrical stimulation of the parafascicular nucleus on the neuronal activities of the subthalamic nucleus and the ventromedial nucleus in rats / 生理学报

The present study was designed to investigate the function and mechanism of high-frequency stimulation (HFS) of the parafascicular nucleus (PF) used as a therapeutic approach for Parkinson's disease (PD). PD rat model was built by injecting 6-hydroxydopamine (6-OHDA) into the substartia nigra pars compacta of adult male Sprague-Dawley rats. Using the ethological methods, we examined the effect of electrical stimulation of PF on the apomorphine-induced rotational behavior in PD rats. Moreover, Electrophysiological recordings were made in rats to investigate the effects of electrical stimulation of PF on the neuronal activities of the subthalamic nucleus (STN) and the ventromedial nucleus (VM). Our results showed that one week after HFS (130 Hz, 0.4 mA, 5 s) of PF, there was significant improvement in apomorphine-induced rotational behavior in PD rats. HFS of PF caused an inhibition of the majority of neurons (84%) recorded in the STN in PD rats. The majority of cells recorded in the VM of the thalamus responded to the HFS with an increase in their unitary discharge activity (81%). These effects were in a frequency-dependent manner. Only stimulus frequencies above 50 Hz were effective. Furthermore, employing microelectrophoresis, we demonstrated that glutamatergic and GABAergic afferent nerve fibers converged on the same STN neurons. These results show that the HFS of PF induces a reduction of the excitatory glutamatergic output from the PF which in turn results in deactivation of STN neurons. The reduction in tonic inhibitory drive from the basal ganglia induces a disinhibition of activity in the VM, a motor thalamic nucleus. In conclusion, the results suggest that HFS of PF may produce a therapeutic effect in PD rats, which is mediated by the nuclei of PF, STN and VM.

Current surgical treatment of Parkinson's disease

There has been a renaissance in the surgical management of Parkinson's disease. This has been due to long-term effects of levodopa and a better understanding of the basal ganglia and its circuitry. Ablative surgery and neurostimulation are the only realistic surgical options at present. Although surgical treatments, such as ablation and stimulation are effective, they are not useful for stopping the progression or restoring the system. Neural transplantation helps restore the system by using a number of techniques. Targets mostly used are in the thalamus, globus pallidus and subthalamic nucleus. A number of factors must be considered including patient's age, disability and his wishes. Globus pallidus stimulation might be preferable for patients who suffer from dyskinesia as a major source of disability. Pallidotomy might be appropriate in cases where frequent stimulator adjustments are impractical. Subthalamic nucleus stimulation is more suitable for patients with significant off periods and in younger patients in whom it may be desirable to maintain intact circuitry. Fetal neural transplantation, stem cell transplantation, xenotransplantation, adrenal medullary transplantation and transplantation of genetically engineered cells are at various stages of development and research. Ethical issues surrounding these process are likely to arouse strong emotions and have to be carefully considered

Estimulação cerebral contínua (DBS) talâ­mica para controle do tremor / Deep brain stimulation of thalamus for tremor control

OBJETIVO: Apresentamos resultados da estimulação contínua do núcleo ventral intermédio (VIM) talâ­mico para o controle do tremor. MÉTODO: Quatro pacientes foram selecionados no período de outubro de 1999 a janeiro de 2001 com tremor incapacitante refratário àáfarmacoterapia. Dois pacientes apresentavam tremor essencial (TE) bilateral e 2 pacientes tremor de repouso por doençaáde Parkinson (DP), um àádireita e outro àáesquerda. Apósáavaliação sistemática, foram submetidos ao implante de eletrodo talâmico, modelo DBS 3387(Medtronic), para estimulação cerebral profunda (ECP) com alta frequência, sendo este bilateral nos casos de TE e unilateral nos casos com tremor por DP. RESULTADOS: Os pacientes tiveram seu seguimento clínico atéáo presente, com média de 12 meses, sendo observada a eficácia da estimulação do núcleo VIM no controle dos disparos das células do tremor. As complicações temporárias do tipo parestesias, disartrias e discreto aumento do tônus foram revertidas após o ajuste dos parâ­meros de estimulação. CONCLUSÄO: Os resultados confirmam os achados da literatura, de que a estimulação talâ­mia éáexcelente opção terapêutica no tratamento do tremor, havendo possibilidade de estimulação talâmica bilateral simultânea com segurança.á

Immunocytochemical Localization of Transforming Growth Factor-alpha in the Forebrain of the Rat / 대한해부학회지

The distribution of Transforming growth factor-alpha (TGF-alpha) was examined in the rat forebrain by immunocytochemistry. TGF-alpha immunoreactivity was observed in the cerebral hemispheres, thalamus and hypothalamus. Neurons in the olfactory and septal area, cerebral cortex, hippocampus, striatum, amygdala, and different nuclei of the thalamus and hypothalamus showed immunoreactivity. The intensity of the immunoreaction was high in the hippocampus, pyramidal cell layers of cerebral cortex, reticular and ventral thalamic nuclei, and paraventricular and supraoptic hypothalamic nuclei. In addition, a few labelled glial cells appeared at random in the forebrain. These results indicate that both neurons and glial cells appear to synthesize TGF-alpha in normal forebrain of the rat. However, TGF-alpha immunoreactivity was more widely distributed in neurons than glial cells. Therefore, although the role of TGF-alpha in the central nervous system remains elusive, the present data support the concept that TGF-alpha may act as a trophic factor in the adult rat forebrain.

The Changes of c-fos and c-jun after Capsaicine Treatment in the Rat Brain / 대한해부학회지

The expression of c-fos and c-jun in the brain of the rat after capsaicin treatment was investigated by in situ hybridization, dot blot hybridization and immunocytochemical methods. Adult male Sprague-Dawley rats[200g] were used for this study. The first set of rats received a single subcutaneous injection of capsaicin[50mg/Kg] dissolved in 10% Tween-80 and 10% ethanol in saline. The rats were decapitated 1, 3, 5, 10, 24, 72 hours and 1 week after capsaicin treatment. The control set of rats were treated with saline instead of capsaicin. In situ hybridization and dot blot hybridization were carried out. O1igonucleotide probe complimentary to c-fos mRNA sequences were used for this study and labeling of oligonucleotides was accomplished using the DNA tailing kit. The expression of c-fos mRNA on the nucleus of neurons in in situ hybridization was observed throughout the brain, and was especially abundant in the olfactory cortex, nucleus of diagonal band of Broca, habenular nuclei, periaqueductal gray, parabrachial nucleus, entopeduncular nucleus, ventral posterolateral nucleus of the thalamus and cerebellum. Compared to the control rats, c-fos mRNA were increased 24 hours after capsaicin injection and gradually decreased after 72 hours, returning to the normal control level 1 week after capsaicin injection. c-fos mRNA was detected only 1 week after capsaicin injection in the various areas of the brain. The fos protein-like immunoreactivity was initially somewhat decreased at 24 hours, but increased at 72 hours and reactions was maximally observed at 1 week after capsaicin treatment. But Jun protein immunoreactivity was not increased, on the contrary, it was even decreased both in numbers of reactive cells and immunoreactivity 1 week after capsaicin injection. From the above results, c-fos gene expression was pronounced in the nucleus concerned with pain, olfaction and taste such as VPL nucleus of the thalamus, olfactory cortex and parabrachial nucleus, in the limbic system concerned with stress and emotion such as nucleus of diagonal band of Broca, periaqueductal gray and habenular nucleus, in the structure concerned with somatic motor function such as entopeduncular nucleus and cerebellum. Also, the c-fos gene was activated by the capsaicin early in the course of effects, then the fos protein increased as a results of c-fos activation. On the other hand, c-jun did not respond to capsaicin treatment early in the course, but Jun protein decreased late in the course of capsaicin effects.