Bingbing Liu

Confocal laser scanning microscopy and ultrastructural study of VGLUT2 thalamic input to striatal projection neurons in rats

We examined thalamic input to striatum in rats using immunolabeling for the vesicular glutamate transporter (VGLUT2). Double immunofluorescence viewed with confocal laser scanning microscopy (CLSM) revealed that VGLUT2+ terminals are distinct from VGLUT1+ terminals. CLSM of Phaseolus vulgaris‐leucoagglutinin (PHAL)‐labeled cortical or thalamic terminals revealed that VGLUT2 is rare in corticostriatal terminals but nearly always present in thalamostriatal terminals. Electron microscopy revealed that VGLUT2+ terminals made up 39.4% of excitatory terminals in striatum (with VGLUT1+ corticostriatal terminals constituting the rest), and 66.8% of VGLUT2+ terminals synapsed on spines and the remainder on dendrites. VGLUT2+ axospinous terminals had a mean diameter of 0.624 μm, while VGLUT2+ axodendritic terminals a mean diameter of 0.698 μm. In tissue in which we simultaneously immunolabeled thalamostriatal terminals for VGLUT2 and striatal neurons for D1 (with about half of spines immunolabeled for D1), 54.6% of VGLUT2+ terminals targeted D1+ spines (i.e., direct pathway striatal neurons), and 37.3% of D1+ spines received VGLUT2+ synaptic contacts. By contrast, 45.4% of VGLUT2+ terminals targeted D1‐negative spines (i.e., indirect pathway striatal neurons), and only 25.8% of D1‐negative spines received VGLUT2+ synaptic contacts. Similarly, among VGLUT2+ axodendritic synaptic terminals, 59.1% contacted D1+ dendrites, and 40.9% contacted D1‐negative dendrites. VGLUT2+ terminals on D1+ spines and dendrites tended to be slightly smaller than those on D1‐negative spines and dendrites. Thus, thalamostriatal terminals contact both direct and indirect pathway striatal neurons, with a slight preference for direct. These results are consistent with physiological studies indicating slightly different effects of thalamic input on the two types of striatal projection neurons. J. Comp. Neurol., 521:1354–1377, 2013.

Melatonin Ameliorates Injury and Specific Responses of Ischemic Striatal Neurons in Rats

Studies have confirmed that middle cerebral artery occlusion (MCAO) causes striatal injury in which oxidative stress is involved in the pathological mechanism. Increasing evidence suggests that melatonin may have a neuroprotective effect on cerebral ischemic damage. This study aimed to examine the morphological changes of different striatal neuron types and the effect of melatonin on striatal injury by MCAO. The results showed that MCAO induced striatum-related dysfunctions of locomotion, coordination, and cognition, which were remarkably relieved with melatonin treatment. MCAO induced severe striatal neuronal apoptosis and loss, which was significantly decreased with melatonin treatment. Within the outer zone of the infarct, the number of Darpp-32+ projection neurons and the densities of dopamine-receptor-1 (D1)+ and dopamine-receptor-2 (D2)+ fibers were reduced; however, both parvalbumin (Parv)+ and choline acetyltransferase (ChAT)+ interneurons were not significantly decreased in number, and neuropeptide Y (NPY)+ and calretinin (Cr)+ interneurons were even increased. With melatonin treatment, the loss of projection neurons and characteristic responses of interneurons were notably attenuated. The present study demonstrates that the projection neurons are rather vulnerable to ischemic damage, whereas the interneurons display resistance and even hyperplasia against injury. In addition, melatonin alleviates striatal dysfunction, neuronal loss, and morphological transformation of interneurons resulting from cerebral ischemia.

Protective effect of melatonin on 3-NP induced striatal interneuron injury in rats.

To confirm the effect of melatonin on 3-nitropropionic acid (3-NP)-induced striatal interneuron injury in rats, behavioral test, histology, immunohistochemistry and Western blotting were respectively used to characterize the behavioral changes of experimental animals in motor and cognition, the morphological changes of striatal interneurons and the expression level of protein markers induced by 3-NP. The results showed that (1) 3-NP induced dysfunction of experimental animals in movement, motor coordination and cognition could be relieved by melatonin treatment; (2) The 3-NP-induced lesion area was unvaryingly in dorsolateral striatum, with almost all neuronal loss in the lesion core, however, lots of neurons survived after melatonin treatment; (3) Immunohistochemical staining of the four interneuron types (parvalbuminergic, cholinergic, calretinergic, and neuropeptide Y-neuronal nitric oxide synthase co-containing) showed that, in the lesion core of 3-NP group, loss of the four interneuron types was obvious, but in transition zone, the processes and varicosities of calretinergic, and neuropeptide Y-neuronal nitric oxide synthase co-containing interneurons increased significantly. Melatonin treatment reduced the loss of the four interneuron types in the lesion core, and inhibited the increase of processes and varicosities in the transition zone; (4) Consistent with above results, the expression level of five interneuron protein markers were significantly increased in the striatum after melatonin treatment. Notably, in both the transition zone and the lesion core induced by 3-NP, TUNEL-positive cells were detected, but decreased significantly after melatonin treatment. The present results indicate that melatonin effectively protects the striatal neurons against the injury induced by 3-NP in rats.

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.

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.

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.

Dihydromyricetin Ameliorates 3NP-induced Behavioral Deficits and Striatal Injury in Rats

Oxidative stress is closely involved in neurodegenerative diseases. The present study aimed to examine the effect of anti-oxidant DHM (dihydromyricetin) on 3NP (3-nitropropionic acid) -induced behavioral deficits of experimental rats and striatal histopathological injury by using behavioral, imaging, biochemistry, histochemistry and molecular biology technologies. The experimental results showed that both motor dysfunctions and learning and memory impairments induced by 3NP were significantly reduced after DHM treatment. 3NP-induced striatal metabolic abnormality was also remarkably improved by DHM treatment, showed as the increased glucose metabolism in PET/CT scan, decreased MDA (malondialdehyde) and increased SOD (superoxide dismutase) activity in enzyme histochemical staining. In addition, the cell apoptosis was evidently detected in the striatum of the 3NP group, while in the 3NP + DHM group, the number of apoptotic cells was remarkably reduced. 3NP treatment obviously induced down-regulation of Bcl-2, and up-regulations of Bax and Cleaved Caspase-3, while these changes were significantly reversed by DHM treatment. The present results suggested that DHM showed its protective effect by anti-oxidant and anti-apoptosis mechanisms.

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.

Cortical regulation of striatal projection neurons and interneurons in a Parkinson's disease rat model

Striatal neurons can be either projection neurons or interneurons, with each type exhibiting distinct susceptibility to various types of brain damage. In this study, 6-hydroxydopamine was injected into the right medial forebrain bundle to induce dopamine depletion, and/or ibotenic acid was injected into the M1 cortex to induce motor cortex lesions. Immunohistochemistry and western blot assay showed that dopaminergic depletion results in significant loss of striatal projection neurons marked by dopamine- and cyclic adenosine monophosphate-regulated phosphoprotein, molecular weight 32 kDa, calbindin, and μ-opioid receptor, while cortical lesions reversed these pathological changes. After dopaminergic deletion, the number of neuropeptide Y-positive striatal interneurons markedly increased, which was also inhibited by cortical lesioning. No noticeable change in the number of parvalbumin-positive interneurons was found in 6-hydroxydopamine-treated rats. Striatal projection neurons and interneurons show different susceptibility to dopaminergic depletion. Further, cortical lesions inhibit striatal dysfunction and damage induced by 6-hydroxydopamine, which provides a new possibility for clinical treatment of Parkinsons disease.