Significance of the epidermal growth factor receptor mutation status and differences among molecular subgroups in surgically resected lung microinvasive adenocarcinoma.

Lung microinvasive adenocarcinoma (MIA) is a newly-defined subtype of early stage non-small cell lung cancer (NSCLC). However, its epidermal growth factor receptor (EGFR) mutation status and clinical significance remain unclear. The present study aimed to determine EGFR mutation characteristics and identify their significance in patients with resected lung MIA. The present study also analyzed clinicopathological differences between EGFR molecular subgroups defined as 19Del and L858R. The present study examined EGFR mutations in 79 consecutive lung MIA resection specimens and compared the differences in clinicopathological features between the EGFR wild-type and mutation groups, as well as between the 19Del and L858R subgroups. EGFR mutations were detected in 60 (75.95%) tumors. The most common mutations were 19Del (28 cases; 35.44%) and L858R (30 cases; 37.97%). Two patients harbored rare mutations and one of them had a concomitant double mutation. EGFR mutations were significantly associated with microinvasion component, thyroid transcription factor 1 (TTF-1) expression, intratumoral fibrosis and inflammatory cell infiltration. Subgroup evaluation indicated that there was a significant association between 19Del and tumor size, maximum diameter of microinvasion, presence of intratumoral fibrosis and inflammatory cell infiltration. Similar associations were observed for the L858R subgroup, and L858R was associated with TTF-1 expression. In particular, 19Del occurred more frequently in MIA with a smaller size, with a smaller microinvasive area, without TTF-1 expression, and lacking intratumoral fibrosis and inflammatory cell infiltration. By contrast, L858R was detected more frequently in MIA with entirely different tumor features. In conclusion, the results of the present study indicated that surgically resected MIA cases harboring different EGFR gene statuses exhibit distinct clinicopathological features. Significant differences in pathological features associated with the tumor microenvironment were identified in MIA with 19Del or L858R mutations. Therefore, the present study proposed that MIA should be classified into molecular subgroups based on EGFR mutation subtypes. The molecular sub-classification should be taken into account for prognostic evaluation and clinical management of MIA.

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.

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.

The effects of unilateral 6-OHDA lesion in medial forebrain bundle on the motor, cognitive dysfunctions and vulnerability of different striatal interneuron types in rats.

In this study, the motor deficit, cognition impairment and the vulnerability of different striatal interneurons to the 6-hydroxydopamine (6-OHDA)-induced excitotoxicity in unilateral medial forebrain bundle (MFB) lesion rats were analyzed by employing behavioral test, immunohistochemistry and Western blot methods. The apomorphine-induced rotation after MFB lesion was used as a valid criterion of motor deficit. The 6-OHDA damaged rats had limb rigidity with longer hang time compared to the controls in the grip strength test. Cognitive and mnemonic deficits of rats with unilateral MFB lesion were observed by the water maze task. The MFB lesion resulted in a significant loss of tyrosine hydroxylase (TH)+ cells in the contralateral striatum or substantia nigra. After dopaminergic depletion, the numbers of calretinin (Cr)+ and choline acetyltransferase (ChAT)+ interneurons were notably reduced while these of neuropeptide Y (NPY)+ were markedly increased in the striatum. No noticeable change in the number of parvalbumin (Parv)+ interneurons was found in 6-OHDA rats. In addition, the fiber densities for each individual interneuron were increased after 6-OHDA treatment, especially for the fiber densities of Parv+ and Cr+ interneurons. The Western blot analysis further confirmed the results described above. In conclusion, the MFB lesion model is suitable to mimic Parkinson's disease (PD), and our results are helpful for further understanding the underlying mechanism and the specific functions of various striatal interneurons in the pathological process of PD.

EPO-dependent activation of PI3K/Akt/FoxO3a signalling mediates neuroprotection in in vitro and in vivo models of Parkinson's disease.

Erythropoietin (EPO) may become a potential therapeutic candidate for the treatment of the neurodegenerative disorder -- Parkinson's disease (PD), since EPO has been found to prevent neuron apoptosis through the activation of cell survival signalling. However, the underlying mechanisms of how EPO exerts its neuroprotective effect are not fully elucidated. Here we investigated the mechanism by which EPO suppressed 6-hydroxydopamine (6-OHDA)-induced neuron death in in vitro and in vivo models of PD. EPO knockdown conferred 6-OHDA-induced cytotoxicity. This effect was reversed by EPO administration. Treatment of PC12 cells with EPO greatly diminished the toxicity induced by 6-OHDA in a dose- and time-dependent manner. EPO effectively reduced apoptosis of striatal neurons and induced a significant improvement on the neurological function score in the rat models of PD. Furthermore, EPO increased the expression of phosphorylated Akt and phosphorylated FoxO3a, and abrogated the 6-OHDA-induced dysregulation of Bcl-2, Bax and Caspase-3 in PC12 cells and in striatal neurons. Meanwhile, the EPO-dependent neuroprotection was notably reversed by pretreatment with LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). Our data suggest that PI3K/Akt/FoxO3a signalling pathway may be a possible mechanism involved in the neuroprotective effect of EPO in PD.

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.

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.

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.

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.

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.