The pathological involvement of spinal cord EphB2 in visceral sensitization in male rats.

Patients with post-traumatic stress disorder (PTSD) are usually at an increased risk for chronic disorders, such as irritable bowel syndrome (IBS), characterized by hyperalgesia and allodynia, but its subsequent effect on visceral hyperalgesia and the mechanism remain unclear. The present study employed single prolonged stress (SPS), a model of PTSD-pain comorbidity, behavioral evaluation, intrathecal drug delivery, immunohistochemistry, Western blotting, and RT-PCR techniques. When detecting visceral sensitivity, the score of the abdominal withdrawal reflex (AWR) induced by graded colorectal distention (CRD) was used. The AWR score was reduced in the SPS day 1 group but increased in the SPS day 7 and SPS day 14 groups at 40 mmHg and 60 mmHg, and the score was increased significantly with EphrinB1-Fc administration. The EphB2+ cell density and EphB2 protein and mRNA levels were downregulated in the SPS day 1 group and then upregulated significantly in the SPS day 7 group; these changes were more noticeable with EphrinB1-Fc administration compared with the SPS-only group. The C-Fos-positive reaction induced by SPS was mainly localized in neurons of the spinal dorsal horn, in which the C-Fos-positive cell density and its protein and mRNA levels were upregulated on SPS days 7 and 14; these changes were statistically significant in the SPS + EphrinB1-Fc group compared with the SPS alone group. The present study confirmed the time window for the AWR value, EphB2 and C-Fos changes, and the effect of EphrinB1-Fc on these changes, which suggests that spinal cord EphB2 activation exacerbates visceral pain after SPS.

Morphological Study of the Cortical and Thalamic Glutamatergic Synaptic Inputs of Striatal Parvalbumin Interneurons in Rats.

Parvalbumin-immunoreactive (Parv+) interneurons is an important component of striatal GABAergic microcircuits, which receive excitatory inputs from the cortex and thalamus, and then target striatal projection neurons. The present study aimed to examine ultrastructural synaptic connection features of Parv+ neruons with cortical and thalamic input, and striatal projection neurons by using immuno-electron microscopy (immuno-EM) and immunofluorescence techniques. Our results showed that both Parv+ somas and dendrites received numerous asymmetric synaptic inputs, and Parv+ terminals formed symmetric synapses with Parv- somas, dendrites and spine bases. Most interestingly, spine bases targeted by Parv+ terminals simultaneously received excitatory inputs at their heads. Electrical stimulation of the motor cortex (M1) induced higher proportion of striatal Parv+ neurons express c-Jun than stimulation of the parafascicular nucleus (PFN), and indicated that cortical- and thalamic-inputs differentially modulate Parv+ neurons. Consistent with that, both Parv + soma and dendrites received more VGlut1+ than VGlut2+ terminals. However, the proportion of VGlut1+ terminal targeting onto Parv+ proximal and distal dendrites was not different, but VGlut2+ terminals tended to target Parv+ somas and proximal dendrites than distal dendrites. These functional and morphological results suggested excitatory cortical and thalamic glutamatergic inputs differently modulate Parv+ interneurons, which provided inhibition inputs onto striatal projection neurons. To maintain the balance between the cortex and thalamus onto Parv+ interneurons may be an important therapeutic target for neurological disorders.

Increase in Glutamatergic Terminals in the Striatum Following Dopamine Depletion in a Rat Model of Parkinson's Disease.

Dopaminergic neuron degeneration is known to give rise to dendrite injury and spine loss of striatal neurons, however, changes of intrastriatal glutamatergic terminals and their synapses after 6-hydroxydopamine (6OHDA)-induced dopamine (DA)-depletion remains controversial. To confirm the effect of striatal DA-depletion on the morphology and protein levels of corticostriatal and thalamostriatal glutamatergic terminals and synapses, immunohistochemistry, immuno-electron microscope (EM), western blotting techniques were performed on Parkinson's disease rat models in this study. The experimental results of this study showed that: (1) 6OHDA-induced DA-depletion resulted in a remarkable increase of Vesicular glutamate transporter 1 (VGlut1) + and Vesicular glutamate transporter 2 (VGlut2)+ terminal densities at both the light microscope (LM) and EM levels, and VGlut1+ and VGlut2+ terminal sizes were shown to be enlarged by immuno-EM; (2) Striatal DA-depletion resulted in a decrease in both the total and axospinous terminal fractions of VGlut1+ terminals, but the axodendritic terminal fraction was not significantly different from the control group. However, total, axospinous and axodendritic terminal fractions for VGlut2+ terminals declined significantly after striatal DA-depletion. (3) Western blotting data showed that striatal DA-depletion up-regulated the expression levels of the VGlut1 and VGlut2 proteins. These results suggest that 6OHDA-induced DA-depletion affects corticostriatal and thalamostriatal glutamatergic synaptic inputs, which are involved in the pathological process of striatal neuron injury induced by DA-depletion.

Neuronal Apoptosis: Pathological Basis of Behavioral Dysfunctions Induced by Angiostrongylus cantonensis in Rodents Model.

Angiostrongylus cantonensis invades the central nervous system (CNS) of humans to induce eosinophilic meningitis and meningoencephalitis and leads to persistent headache, cognitive dysfunction, and ataxic gait. Infected mice (nonpermissive host), admittedly, suffer more serious pathological injuries than rats (permissive host). However, the pathological basis of these manifestations is incompletely elucidated. In this study, the behavioral test, histological and immunohistochemical techniques, and analysis of apoptotic gene expression, especially caspase-3, were conducted. The movement and motor coordination were investigated at week 2 post infection (PI) and week 3 PI in mice and rats, respectively. The cognitive impairs could be found in mice at week 2 PI but not in rats. The plaque-like lesion, perivascular cuffing of inflammatory cells, and dilated vessels within the cerebral cortex and hippocampus were more serious in mice than in rats at week 3 PI. Transcriptomic analysis showed activated extrinsic apoptotic pathway through increased expression of TNFR1 and caspase-8 in mice CNS. Immunohistochemical and double-labeling for NeuN and caspase-3 indicated the dramatically increased expression of caspase-3 in neuron of the cerebral cortex and hippocampus in mice but not in rats. Furthermore, western-blotting results showed high expression of cleaved caspase-3 proteins in mice but relatively low expression in rats. Thus, extrinsic apoptotic pathway participated in neuronal apoptosis might be the pathological basis of distinct behavioral dysfunctions in rodents with A. cantonensis infection. It provides the evidences of a primary molecular mechanism for the behavioral dysfunction and paves the ways to clinical diagnosis and therapy for A. cantonensis infection.

Characteristic Changes of Astrocyte and Microglia in Rat Striatum Induced by 3-NP and MCAO.

Our previous studies had confirmed that both 3-NP and MCAO induced the behavioral defect as well as striatal neuronal injury and loss in experimental rats. This study aimed to examine different response forms of striatal astrocyte and microglia in 3-NP and MCAO rat models. The present results showed that the immunoreaction for GFAP was extremely weak in the lesioned core of striatum, but in the transition zone of 3-NP model and the penumbra zone of MCAO model, GFAP+ cells showed strong hypertrophic and proliferative changes. Statistical analysis for the number, size and integral optical density (IOD) of GFAP+ cells showed significant differences when compared with their controls and compared between the core and the transition zone or the penumbra zone, respectively, but no differences between the 3-NP and MCAO groups. However, Iba-1+ cells showed obvious hypertrophy and proliferation in the injured striatum in the 3-NP and the MCAO models, especially in the transition zone of 3-NP model and the penumbra zone of MCAO model. These Iba-1+ cells displayed two characteristic forms as branching cells with thick processes and amoeboid cells with thin processes. Statistical analysis showed that the number, size and IOD of Iba-1+ cells were significantly increased in the cores and the transition zone of 3-NP group and the penumbra zone of MCAO group than that of the controls, and the immune response of Iba-1 was stronger in the MCAO group than in the 3-NP group. The present results suggested that characteristic responses of astrocyte and microglia in the 3-NP and the MCAO models display their different effects on the pathological process of brain injury.

Microglia activation: one of the checkpoints in the CNS inflammation caused by Angiostrongylus cantonensis infection in rodent model.

Angiostrongylus cantonensis (A. cantonensis) is a rodent nematode. Adult worms of A. cantonensis live in the pulmonary arteries of rats; humans are non-permissive hosts like the mice. The larva cannot develop into an adult worm and only causes serious eosinophilic meningitis or meningo-encephalitis if humans or mice eat food containing larva of A. cantonensis in the third stage. The differing consequences largely depend on differing immune responses of hosts to parasite during A. cantonensis invasion and development. To further understand the reasons why mice and rats attain different outcomes in A. cantonensis infection, we used the HE staining to observe the pathological changes of infected mice and rats. In addition, we measured mRNA levels of some cytokines (IL-5, IL-6, IL-13, Eotaxin, IL-4, IL-10, TGF-ß, IFN-γ, IL-17A, TNF-α, IL-1ß, and iNOS) in brain tissues of mice and rats by real-time PCR. The result showed that brain inflammation in mice was more serious than in rats. Meanwhile, mRNA expression levels of IL-6, IL-1ß, TNF-α, and iNOS increased after mice were infected. In contrast, mRNA levels of these cytokines in rats brain tissues decreased at post- infection 21 days. These cytokines mostly were secreted by activated microglia in central nervous system. Microglia of mice and rats were showed by Iba-1 (microglia marker) staining. In micee brains, microglia got together and had more significant activation than in rats brains. The results demonstrate that mice and rats have different CNS inflammation after infection by A. cantonensis, and it is in line with other researchers' reported findings. In conclusion, it is suggested that microglia activation is probably to be one of the most important factors in angiostrongyliasis from our study.

Morphological diversity of GABAergic and cholinergic interneurons in the striatal dorsolateral and ventromedial regions of rats.

The striatum plays a fundamental role in sensorimotor and cognitive functions of the body, and different sub-regions control different physiological functions. The striatal interneurons play important roles in the striatal function, yet their specific functions are not clearly elucidated so far. The present study aimed to investigate the morphological properties of the GABAergic interneurons expressing neuropeptide Y (NPY), calretinin (Cr), and parvalbumin (Parv) as well as the cholinergic interneurons expressing choline acetyltransferase (ChAT) in the striatal dorsolateral (DL) and ventromedial (VM) regions of rats using immunohistochemistry and Western blot. The present results showed that the somatic size of Cr+ was the smallest, while ChAT+ was the largest among the four types of interneurons. There was no regional difference in neuronal somatic size of all types of interneurons. Cr+ and Parv+ neurons were differentially distributed in the striatum. Moreover, Parv+ had the longest primary dendrites in the DL region, while NPY+ had the longest ones in the VM region of striatum. But there was regional difference in the length of primary dendrites of Parv. The numbers of primary dendrites of Parv+ were the largest in both DL and VM regions of striatum. Both Cr+ and Parv+ primary dendrites displayed regional difference in the striatum. Western blot further confirmed the regional differences in the protein expression level of Cr and Parv. Hence, the present study indicates that GABAergic and cholinergic interneurons might be involved in different physiological functions based on their morphological and distributional diversity in different regions of the rat striatum.

Melatonin reduces projection neuronal injury induced by 3-nitropropionic acid in the rat striatum.

BACKGROUND: Melatonin has shown a protective effect against various oxidative damages in the nervous system. Our previous studies have also confirmed its effect on behavioral dysfunction of experimental rats and injury of striatal interneurons induced by 3-nitropropionic acid. The present study aimed to further determine the effect of melatonin on the injury of striatal projection neurons induced by 3-nitropropionic acid. METHODS: Classic histology, immunohistochemistry, Western blotting and immunoelectron microscopy were applied in this study. RESULTS: The results were as follows: (1) in the striatum, 3-nitropropionic acid induced a clear lesion area with a transition zone around it, in which both D1+ and D2+ fibers were decreased significantly. However, in the group with melatonin treatment, the striatal lesion area was smaller than in the 3-nitropropionic acid group and the loss of D1+ and D2+ fibers was less pronounced than in the 3-nitropropionic acid group. (2) Histochemical results showed that the dendritic spine density of striatal projection neurons was decreased more seriously after 3-nitropropionic acid treatment, whereas the loss of dendritic spines was less marked in the melatonin-treated group than in the 3- nitropropionic acid group. Immunoelectron microscopy showed that the density of D1+ and D2+ dendrites and spines was significantly decreased in the 3-nitropropionic acid group, and the loss of D1+ and D2+ spines as well as D2+ dendrites was significantly reversed by melatonin administration. (3) Western blotting showed that the expression level of projection neuron protein markers decreased more significantly in the 3-nitropropionic acid group than in the control group and increased significantly in the melatonin-treated group. CONCLUSIONS: The present results suggest that 3-nitropropionic acid induces serious injury of striatal projection neurons and that melatonin effectively protects against this pathological damage.

Morphological changes in perisynaptic astrocytes induced by dopamine neuronal degeneration in the striatum of rats.

Introduction: The typical functionality of astrocytes was previously shown to be disrupted by Parkinson's disease (PD), which actively regulates synaptic neurotransmission. However, the morphological changes in astrocytes wrapping glutamatergic synapses in the striatum after dopamine (DA) neuronal degeneration is unclear. Methods: We utilized a range of methodologies, encompassing the 6-hydroxydopamine (6OHDA)-induced PD model, as well as techniques such as immunohistochemistry, Western blotting, immunofluorescence and immunoelectron microscopy (IEM) to delve into the consequences of DA neuronal degeneration on the morphological attributes of perisynaptic astrocytes. Results: Our findings demonstrated a notable rise in glial fibrillary acidic protein (GFAP) + astrocyte density and an upregulation in GFAP protein expression within the striatum due to DA neuronal degeneration, coincided with the enlargement, elongation, and thickening of astrocyte protuberances. However, the expression levels of glutamate transporter 1 (GLT1) and glutamine synthetase (GS), which are related to glutamate-glutamine cycle, were significantly reduced. Double immunofluorescence and IEM results indicated that different proportions of vesicular glutamate transporter 1 (VGlut1)+ and vesicular glutamate transporter 2 (VGlut2) + terminals were wrapped by astrocytes. Additionally, DA neuronal degeneration increased the percentage and area of VGlut1+ and VGlut2+ terminals wrapped by GFAP + astrocytes in the striatum. Furthermore, we noted that DA neuronal degeneration increased the percentage of VGlut1+ and VGlut2+ axo-spinous synapses wrapped by astrocytes but had no effect on axo-dendritic synapses. Conclusion: Hence, perisynaptic astrocytes wrapping striatal glutamatergic synapses exhibit substantial morphological and functional alterations following DA neuronal degeneration making them a potential target for therapeutic interventions in PD.

A neural tract tracing study on synaptic connections for cortical glutamatergic terminals and cervical spinal calretinin neurons in rats.

The cerebral cortex innervates motor neurons in the anterior horn of the spinal cord by regulating of interneurons. At present, nerve tracing, immunohistochemistry, and immunoelectron microscopy are used to explore and confirm the characteristics of synaptic connections between the corticospinal tract (CST) and cervical spinal calretinin (Cr) interneurons. Our morphological results revealed that (1) biotinylated dextran amine labeled (BDA+) fibers from the cerebral cortex primarily presented a contralateral spinal distribution, with a denser distribution in the ventral horn (VH) than in the dorsal horn (DH). An electron microscope (EM) showed that BDA+ terminals formed asymmetric synapses with spinal neurons, and their mean labeling rate was not different between the DH and VH. (2) Cr-immunoreactive (Cr+) neurons were unevenly distributed throughout the spinal gray matter, and were denser and larger in the VH than in the DH. At the single labeling electron microscope (EM) level, the labeling rate of Cr+ dendrites was higher in the VH than in the DH, in which Cr+ dendrites mainly received asymmetric synaptic inputs, and between the VH and DH. (3) Immunofluorescence triple labeling showed obvious apposition points among BDA+ terminals, synaptophysin and Cr+ dendrites, with a higher density in the VH than in the DH. (4) Double labeling in EM, BDA+ terminals and Cr+ dendrites presented the same pattern, BDA+ terminals formed asymmetric synapses either with Cr+ dendrites or Cr negative (Cr-) dendrites, and Cr+ dendrites received either BDA+ terminals or BDA- synaptic inputs. The average percentage of BDA+ terminals targeting Cr+ dendrites was higher in the VH than in the DH, but the percentage of BDA+ terminals targeting Cr- dendrites was prominently higher than that targeting Cr+ dendrites. There was no difference in BDA+ terminal size. The percentage rate for Cr+ dendrites receiving BDA+ terminal inputs was lower than that receiving BDA- terminal inputs, and the BDA+ terminal size was larger than the BDA- terminal size received by Cr+ dendrites. The present morphological results suggested that spinal Cr+ interneurons are involved in the regulatory process of the cortico-spinal pathway.

Intestinal ischemia/reperfusion enhances microglial activation and induces cerebral injury and memory dysfunction in rats.

OBJECTIVE: The mortality of critically ill patients associated with intestinal ischemia/reperfusion remains very high, which results from multiorgan dysfunction or failure due to intestinal injury induced by intestinal ischemia/reperfusion. This study was carried out to investigate whether intestinal ischemia/reperfusion can cause cerebral injury and concomitant memory dysfunction, and explore the potential mechanisms. DESIGN: Prospective, controlled, and randomized animal study. SETTING: University research laboratory. SUBJECTS: Male, adult Sprague-Dawley rats (weighing 250-300 g). INTERVENTIONS: Intestinal ischemia/reperfusion was established by clamping the superior mesenteric artery for 90 mins followed by different reperfusion durations (2, 6, 12, 24, or 48 hrs). The sham surgical preparation including isolation of the superior mesenteric artery without occlusion was performed as control. MEASUREMENTS AND MAIN RESULTS: In comparison with sham control, intestinal ischemia/reperfusion caused severe intestinal injury, accompanied by notable cerebral damage evidenced by increased wet-to-dry brain weight ratio reflecting brain edema and neuronal cell apoptosis manifested by increased apoptotic cell number and cleaved caspase-3 protein expressions. All these changes were concomitant with reduced survival rates as well as impaired memory function determined by Morris water maze test at 24 and 48 hrs after reperfusion. In addition, intestinal ischemia/reperfusion resulted in significant increases in the levels of tumor necrosis factor-α and interleukin-6 both in the serum and in cortices and hippocampal Cornu Ammonis area 1 regions, concomitant with the activation of microglia, a key cellular mediator involved in neuroinflammation and neurodegeneration, which was evidenced by increased protein expressions of ionized calcium binding adaptor molecule 1. Furthermore, the releases of reactive oxygen species evidenced by increased malondialdehyde levels and decreased superoxide dismutase activities in cortices and hippocampal Cornu Ammonis area 1 regions were found after reperfusion. CONCLUSIONS: These findings indicate that intestinal ischemia/reperfusion-induced intestinal injury can lead to cerebral damage and memory dysfunction partly via microglia activation which further facilitates oxidative injury, inflammatory response, and neuronal cell apoptosis.

Differences of larval development and pathological changes in permissive and nonpermissive rodent hosts for Angiostrongylus cantonensis infection.

Angiostrongylus cantonensis is a neurotrophic and pulmonary parasite which causes severe neuropathological damages by invading and developing in the central nervous system (CNS). Nonpermissive host with A. cantonensis infection appeared to have more serious neurologic symptoms, and there is still not much knowledge about the host-parasite interrelationship in different hosts. We investigated and compared the larval size, recovery rate, distribution, and the severity of pathologic injuries in the CNS of both permissive host (e.g., rats) and nonpermissive hosts (e.g., mice). In present study, mice infected with A. cantonensis showed higher worm recovery rate in late-stage infection and smaller size of intracranial larvae as compared to the infected rats. Intracranial larvae mainly aggregated on cerebral surface of infected rats but on surface of cerebellum and brainstem in mice. Hemorrhage and tissue edema on brain surface caused by worm migration appeared earlier and severer in infected mice than in rats. Neuropathological examination revealed that injuries induced by A. cantonensis in brain parenchyma included hemorrhage, vascular dilatation, focal necrosis with neuronal loss, and infiltration of inflammatory cells. In the comparison of these pathological changes in rats and mice, infected mice suffered more serious injuries and provoked more intense inflammatory response as compared to infected rats. All these morphological evidences indicate that larval development was retardant in the CNS of nonpermissive host, and nonpermissive host experienced more serious pathological injuries than permissive host. It implies that the difference in innate immune response to parasite infection attribute to host specificity.

Preferential interneuron survival in the transition zone of 3-NP-induced striatal injury in rats.

Histology, immunohistochemistry, and Western blotting were used to characterize the changes in morphology, distribution pattern, and marker protein expression of striatal interneurons in the transition zone of striatal injury induced by 3-NP. The 3-NP treatment in rats yielded movement, motor coordination, and cognitive dysfunction. The 3-NP-induced lesion core was unvaryingly in the dorsolateral striatum, with a transition zone of lesser damage around the lesion core, in which medium-sized neurons were significantly decreased in abundance, but larger neurons survived. In both the transition zone and the lesion core, many TUNEL-positive cells negative for the interneuron markers were detected, indicating widespread projection neuron death. Immunohistochemical staining for the four interneuron types (parvalbuminergic, cholinergic, calretinergic, and neuropeptide Y-neuronal nitric oxide synthase cocontaining) showed that few immunolabeled interneurons were observed in the lesion core, but interneuron perikarya showed no evident loss in the transition zone. Consistently with this, Western blotting showed that the five interneuron protein markers were significantly decreased in the striatum after 3-NP treatment. Transition-zone calretinergic and neuropeptide Y-neuronal nitric oxide synthase-cocontaining interneurons, however, possessed more processes and varicosities than normal. These results show that, although striatal interneurons survive in the transition zone after 3-NP-mediated striatal injury, they have enhanced marker protein levels in their processes.

An experimental study on the effect of safflower yellow on tendon injury-repair in chickens.

BACKGROUND: The present study sought to investigate pathologic changes in tendon, expression of basic fibroblast growth factor (bFGF) and collagen type I, and effects of safflower yellow (SY) on the process of tendon injury-repair. MATERIALS AND METHODS: A tendon injury-repair model was used, and stereology, biomechanics, and immunohistochemistry were employed to assess the benefits of local application of SY for the repair. In this model, the flexor digitorum profundus muscle tendon of the third digit was transected bilaterally, and the transected ends sutured. Data were analyzed with SPSS ver. 10.0 software (SPSS Inc., Chicago, IL). RESULTS: The adhesion to surrounding tissues and tensile strength gradually increased after the injury and repair in control (no-SY) tendons, and were significantly greater by the sixth wk than any other time. In the SY tendons, adhesion was significantly lower, and tensile strength significantly higher than in no-SY tendons at the same post-injury-suture time points. An inflammatory reaction was observed in the injury-repair areas of the tendon by the end of first wk post-injury-suture, and reached its peak by the end of second wk. The inflammatory reaction was significantly less in SY tendons than in controls. Immunostaining for bFGF occurred in the tendon injury-repair areas by the end of first wk, and the number of bFGF positive cells reached a peak by the end of second wk, with a greater abundance in SY than control tendons from the second to sixth wk. Expression of collagen type I protein was observed in the injury-repair areas as well, coincident with bFGF, and was remarkably higher in SY than in controls. CONCLUSIONS: Tendon adhesion and tensile strength increased with time post-injury-suture repair, as did expression of bFGF and collagen type I protein in the injured area. SY enhanced expression of bFGF and collagen type I protein, enhanced the tensile strength of the injured tendon, and alleviated the injured tendon adhesion and inflammatory reaction. The results indicated that SY promoted the repair of injured tendon by up-regulating expression of bFGF and collagen type I protein.

The morphological characteristics of corticostriatal and thalamostriatal neurons and their intrastriatal terminals in rats.

PURPOSE: The glutamatergic projection from the cerebral cortex and the thalamus extensively innervates the neostriatal neurons. However, some conflicts in the published literatures about cortical and thalamic intrastriatal synaptic terminals still need to be resolved. The present study intends to further elucidate the morphological characteristics of these two types of the terminals and their neurons. METHODS: The corticostriatal and thalamostriatal terminals were immunolabeled for vesicular glutamate transporter type 1 (VGluT1) and 2 (VGluT2), respectively, and their neurons were retrograde labeled by biotinylated dextran amine 3,000 molecular weight (BDA3k) injection into the dorsolateral striatum of rats. The characteristics of the corticostriatal and thalamostriatal terminals were observed at the LM and EM levels, and the data were statistically analyzed with SPSS10.0 software. RESULTS: We observed that 63.53% of VGluT1+ terminals synapsed on dendritic spines, which was different from VGluT2+ terminals with the equal percentage of synapses on spines and dendrites (14.88 and 17.86%, respectively). Notably, VGluT1+ axospinous synaptic terminals were remarkably larger than VGluT2+ axospinous synaptic terminals. Terminal size-frequency distribution analysis showed that VGluT1+ terminals were within the size ranges of 0.4-0.5 and 0.8-0.9 µm, and VGluT2+ terminals were in the ranges of 0.4-0.5 and 0.6-0.7 µm. Perforated-postsynaptic densities (-PSDs) were more frequently found in VGluT1+ axospinous synaptic terminals than in VGluT2+ axospinous terminals. Furthermore, BDA3k-labeled corticostrital neurons were larger in perikaryal diameter than the thalamostriatal neurons, and they were also categorized as the two main populations based on their size-frequency distribution. CONCLUSIONS: The morphological characteristics of corticostriatal and thalamostriatal terminals and neurons have implications for understanding the roles of synaptic plasticity in adaptive motor control by the basal ganglia, and they have facilitations for understanding the complexities of basal ganglia function.

Characteristic response of striatal astrocytes to dopamine depletion.

Astrocytes and astrocyte-related proteins play important roles in maintaining normal brain function, and also regulate pathological processes in brain diseases and injury. However, the role of astrocytes in the dopamine-depleted striatum remains unclear. A rat model of Parkinson's disease was therefore established by injecting 10 µL 6-hydroxydopamine (2.5 µg/µL) into the right medial forebrain bundle. Immunohistochemical staining was used to detect the immunoreactivity of glial fibrillary acidic protein (GFAP), calcium-binding protein B (S100B), and signal transducer and activator of transcription 3 (STAT3) in the striatum, and to investigate the co-expression of GFAP with S100B and STAT3. Western blot assay was used to measure the protein expression of GFAP, S100B, and STAT3 in the striatum. Results demonstrated that striatal GFAP-immunoreactive cells had an astrocytic appearance under normal conditions, but that dopamine depletion induced a reactive phenotype with obvious morphological changes. The normal striatum also contained S100B and STAT3 expression. S100B-immunoreactive cells were uniform in the striatum, with round bodies and sparse, thin processes. STAT3-immunoreactive cells presented round cell bodies with sparse processes, or were darkly stained with a large cell body. Dopamine deprivation induced by 6-hydroxydopamine significantly enhanced the immunohistochemical positive reaction of S100B and STAT3. Normal striatal astrocytes expressed both S100B and STAT3. Striatal dopamine deprivation increased the number of GFAP/S100B and GFAP/STAT3 double-labeled cells, and increased the protein levels of GFAP, S100B, and STAT3. The present results suggest that morphological changes in astrocytes and changes in expression levels of astrocyte-related proteins are involved in the pathological process of striatal dopamine depletion. The study was approved by Animal Care and Use Committee of Sun Yat-sen University, China (Zhongshan Medical Ethics 2014 No. 23) on September 22, 2014.

Partial decortication ameliorates dopamine depletion­induced striatal neuron lesions in rats.

The balance between glutamate (cortex and thalamus) and dopamine (substantia nigra) inputs on striatal neurons is of vital importance. Dopamine deficiency, which breaks this balance and leads to the domination of cortical glutamatergic inputs, plays an important role in Parkinson's disease (PD). However, the exact impact on striatal neurons has not been fully clarified. Thus, the present study aimed to characterize the influence of corticostriatal glutamatergic inputs on striatal neurons after decortication due to dopamine depletion in rats. 6­Hydroxydopamine was injected into the right medial forebrain bundle to induce dopamine depletion, and/or ibotenic acid into the primary motor cortex to induce decortication. Subsequently, the grip strength test and Morris water maze task indicated that decortication significantly shortened the hang time and the latency that had been increased in the rats subjected to dopamine depletion. Golgi staining and electron microscopy analysis showed that the total dendritic length and dendritic spine density of the striatal neurons were decreased in the dopamine­depleted rats, whereas decortication alleviated this damage. Immunohistochemistry analysis demonstrated that decortication decreased the number of caspase­3­positive neurons in the dopamine­depleted rats. Moreover, reverse transcription­quantitative PCR and western blot analyses showed that decortication offset the upregulation of caspase­3 at both the protein and mRNA levels in the dopamine­depleted rats. In conclusion, the present study demonstrated that a relative excess of cortical glutamate inputs had a substantial impact on the pathological processes of striatal neuron lesions in PD.

Effects of the aqueous extract of the Chinese medicine Danggui-Shaoyao-San on rat pineal melatonin synthesis.

OBJECTIVES: The purpose of this study is to investigate if the aqueous extract of the Chinese medicine Danggui-Shaoyao-San (DSS) can increase the plasma level of melatonin and enhance the function of the pineal gland of naturally aged rats. METHODS: The rats were treated with DSS at doses of 3ml or same volume of distilled water by oral administration at 11 p.m. for three weeks. The plasma level of melatonin were measured by radioimmunoassay. The function of pineal gland were measured through three parameters: pineal beta adrenergic receptor binding investigated by [3H]DHA binding; pineal expression of NAT mRNA detected by real-time RT-PCR; phosphorylation of CREB (P-CREB) and total level of CREB (T-CREB) measured by western blot analysis. RESULTS: DSS significantly increased melatonin level at night after oral administration for 3 weeks. By measurement of pineal [3H]DHA binding, it was found DSS improved the beta-adrenergic receptors binding in pineals. The stimulatory effect of DSS on the expression of NAT mRNA in the old rat pineal gland has been demonstrated in this study. Western blot analysis showed that DSS significantly increased phosphorylation of CREB. CONCLUSIONS: Our results indicate that a downstream pathway for DSS induction of melatonin synthesis in the rat pineal gland acts via cyclic AMP-dependent cascade and transcription mechanism.

A Comparative study for striatal-direct and -indirect pathway neurons to DA depletion-induced lesion in a PD rat model.

Striatal-direct and -indirect Pathway Neurons showed different vulnerability in basal ganglia disorders. Therefore, present study aimed to examine and compare characteristic changes of densities, protein and mRNA levels of soma, dendrites, and spines between striatal-direct and -indirect pathway neurons after DA depletion by using immunohistochemistry, Western blotting, real-time PCR and immunoelectron microscopy techniques. Experimental results showed that: 1) 6OHDA-induced DA depletion decreased the soma density of striatal-direct pathway neurons (SP+), but no significant changes for striatal-indirect pathway neurons (ENK+). 2) DA depletion resulted in a decline of dendrite density for both striatal-direct (D1+) and -indirect (D2+) pathway neurons, and D2+ dendritic density declined more obviously. At the ultrastructure level, the densities of D1+ and D2+ dendritic spines reduced in the 6OHDA groups compared with their control groups, but the density of D2+ dendritic spines reduced more significant than that of D1. 3) Striatal DA depletion down-regulated protein and mRNA expression levels of SP and D1, on the contrary, ENK and D2 protein and mRNA levels of indirect pathway neurons were up-regulated significantly. Present results suggested that indirect pathway neurons be more sensitive to 6OHDA-induced DA depletion.

Activation of RAGE/STAT3 pathway by methylglyoxal contributes to spinal central sensitization and persistent pain induced by bortezomib.

Bortezomib is a first-line chemotherapeutic drug widely used for multiple myeloma and other nonsolid malignancies. Although bortezomib-induced persistent pain is easily diagnosed in clinic, the pathogenic mechanism remains unclear. Here, we studied this issue with use of a rat model of systemic intraperitoneal administration of bortezomib for consecutive 5days. Consisted with our previous study, we found that bortezomib treatment markedly induced mechanical allodynia in rats. Furthermore, we first found that bortezomib treatment significantly induced the upregulation of methylglyoxal in spinal dorsal horn of rats. Spinal local application of methylglyoxal also induced mechanical allodynia and central sensitization in normal rats. Moreover, administration of bortezomib upregulated the expression of receptors for advanced glycation end products (RAGE) and phosphorylated STAT3 (p-STAT3) in dorsal horn. Importantly, intrathecal injection of metformin, a known scavenger of methylglyoxal, significantly attenuated the upregulation of methylglyoxal and RAGE in dorsal horn, central sensitization and mechanical allodynia induced by bortezomib treatment, and blockage of RAGE also prevented the upregulation of p-STAT3, central sensitization and mechanical allodynia induced by bortezomib treatment. In addition, inhibition of STAT3 activity by S3I-201 attenuated bortezomib-induced mechanical allodynia and central sensitization. Local knockdown of STAT3 also ameliorated the mechanical allodynia induced by bortezomib administration. Our results suggest that accumulation of methylglyoxal may activate the RAGE/STAT3 signaling pathway in dorsal horn, and contributes to the spinal central sensitization and persistent pain induced by bortezomib treatment.