Piao Zhang

Erratum to: Injection of Aβ1-40 into hippocampus induced cognitive lesion associated with neuronal apoptosis and multiple gene expressions in the tree shrew

Alzheimer’s disease (AD) can incur significant health care costs to the patient, their families, and society; furthermore, effective treatments are limited, as the mechanisms of AD are not fully understood. This study utilized twelve adult male tree shrews (TS), which were randomly divided into PBS and amyloidbetapeptide1-40 (Aβ1-40) groups. AD model was established via an intracerebroventricular (icv) injection of Aβ1-40 after being incubated for 4 days at 37 °C. Behavioral, pathophysiological and molecular changes were evaluated by hippocampal-dependent tasks, magnetic resonance imaging (MRI), silver staining, hematoxylin–eosin (HE) staining, TUNEL assay and gene sequencing, respectively. At 4 weeks post-injection, as compared with the PBS group, in Aβ1-40 injected animals: cognitive impairments happened, and the hippocampus had atrophied indicated by MRI findings; meanwhile, HE staining showed the cells of the CA3 and DG were significantly thinner and smaller. The average number of cells in the DG, but not the CA3, was also significantly reduced; furthermore, silver staining revealed neurotic plaques and neurofibrillary tangles (NFTs) in the hippocampi; TUNEL assay showed many cells exhibited apoptosis, which was associated with downregulated BCL-2/BCL-XL-associated death promoter (Bad), inhibitor of apoptosis protein (IAP), Cytochrome c (CytC) and upregulated tumor necrosis factor receptor 1 (TNF-R1); lastly, gene sequencing reported a total of 924 mobilized genes, among which 13 of the downregulated and 19 of the upregulated genes were common to the AD pathway. The present study not only established AD models in TS, but also reported on the underlying mechanism involved in neuronal apoptosis associated with multiple gene expression.

Lentiviral-Mediated Netrin-1 Overexpression Improves Motor and Sensory Functions in SCT Rats Associated with SYP and GAP-43 Expressions.

Spinal cord injury (SCI), as a major cause of disability, usually causes serious loss of motor and sensory functions. As a bifunctional axonal guidance cue, netrin-1 can attract axons via the deleted in colorectal cancer (DCC) receptors and repelling others via Unc5 receptors, but its exact role in the recovery of motor and sensory function has not well been studied, and the mechanisms remains elusive. The aim of this experiment is to determine whether lentiviral (LV)-mediated overexpression of netrin-1 or RNA interference (RNAi) can regulate the functional recovery in rats subjected to spinal cord transection (SCT). Firstly, two lentiviral vectors including Lv-exNtn-1 (netrin-1 open reading frame (ORF)) and Lv-shNtn-1 (netrin-1 sh) were constructed and injected into spinal cords rostral and caudal to the transected lesion site. Overexpressing netrin-1 enhanced significantly locomotor function, and reduced thermal and mechanical stimuli in vivo, compared with the control, while silencing netrin-1 did not significantly change the situation. Western blot and immunostaining analysis confirmed that netrin-1 ORF treatment not only effectively increased the expression level of netrin-1, also up-regulated the level of synaptophysin (SYP) in spinal cord rostral to the lesion, but also enhanced growth-associated protein-43 (GAP-43) expression in spinal cord caudal to the lesion site. Comparatively, knockdown of netrin-1 did not give rise to positive findings in our experimental condition. These findings therefore pointed that Lv-mediated netrin-1 overexpression could promote motor and sensory functional recoveries following SCT, and the underlying mechanisms were associated with SYP and GAP-43 expressions. The present study therefore provided a novel strategy for the treatment of SCI and explained the possible mechanisms for the functional improvement.

Interleukin‑6 RNA knockdown ameliorates acute lung injury induced by intestinal ischemia reperfusion in rats by upregulating interleukin‑10 expression

Acute lung injury (ALI) is a common complication following intestinal ischemia/reperfusion (II/R) injury and contributes to the associated high mortality rate. However, the underlying mechanism is poorly understood and treatments are limited. RNA interference (RNAi) has been demonstrated to provide a promising disease treatment strategy both in vitro and in vivo. Therefore, the present study aimed to test whether blocking the proinflammatory cytokine IL-6 by RNAi may protect the lungs from remote organ injury following II/R, and to investigate the potential underlying mechanisms. A total of 176 adult healthy male Sprague-Dawley rats were randomly divided into sham, II/R, negative-control and IL-6-short hairpin (sh)RNA groups. The rats underwent II/R injury with occlusion of the superior mesenteric artery and coeliac artery to induce ischemia for 40 min, and were subsequently reperfused for 0–48 h. The negative-control group received a control lentiviral vector containing scrambled or non-specific sequences, and the IL-6-shRNA groups were administered with a vector containing an IL-6 shRNA sequence to affect RNAi-mediated knockdown of IL-6. ALI severity was determined by lung edema (lung wet/dry ratio) and histological analysis (lung injury scores). IL-6 localization, and mRNA and protein expression levels, were detected by immunofluorescence, reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. IL-10 expression induced by IL-6 knockdown in lung tissues was additionally detected. IL-6 RNAi was revealed to significantly reduce the expression of IL-6, which was associated with upregulated IL-10 expression in lung tissues. Consequently, the severities of ALI and edema induced by II/R were substantially improved. In conclusion, the present study demonstrated that IL-6 RNAi may protect the lung from ALI induced by II/R, and that this protective role may be associated with upregulation of IL-10. These findings may contribute to the development of an IL-6-RNAi-based therapeutic strategy for the treatment of II/R-induced ALI.

Bone Marrow Mesenchymal Stem-Cell Transplantation Promotes Functional Improvement Associated with CNTF-STAT3 Activation after Hemi-Sectioned Spinal Cord Injury in Tree Shrews

Hemi-sectioned spinal cord injury (hSCI) can lead to spastic paralysis on the injured side, as well as flaccid paralysis on the contralateral side, which can negatively affect a patient’s daily life. Stem-cell therapy may offer an effective treatment option for individuals with hSCI. To examine the role of bone marrow mesenchymal stem cells (BMSCs) transplantation on hSCI and explore related mechanisms in the tree shrews, here, we created a model of hSCI by inducing injury at the tenth thoracic vertebra (T10). Hoechst 33342-labeled BMSCs derived from adult tree shrews were isolated, cultured, and implanted into the spinal cord around the injury site at 9 days after injury. The isolated BMSCs were able to survive, proliferate and release a variety of neurotrophic factors (NTFs) both in vitro and in vivo. At 28 days after injury, compared with the sham group, the hSCI group displayed scar formation and dramatic elevations in the mean interleukin 1 beta (IL-1β) density and cell apoptosis level, whereas the expression of signal transducer and activator of transcription 3 (STAT3) and ciliary neurotrophic factor (CNTF) mRNA was reduced. Following BMSC transplantation, motoneurons extent of shrinkage were reduced and the animals’ Basso, Beattie, and Bresnahan (BBB) locomotion scale scores were significantly higher at 21 and 28 days after injury when compared with the injured group. Moreover, the hSCI-induced elevations in scar formation, IL-1β, and cell apoptosis were reduced by BMSC transplantation to levels that were close to those of the sham group. Corresponding elevations in the expression of STAT3 and CNTF mRNA were observed in the hSCI + BMSCs group, and the levels were not significantly different from those observed in the sham group. Together, our results support that grafted BMSCs can significantly improve locomotor function in tree shrews subjected to hSCI and that this improvement is associated with the upregulation of CNTF and STAT3 signaling.

Tree shrew neural stem cell transplantation promotes functional recovery of tree shrews with a hemi‑sectioned spinal cord injury by upregulating nerve growth factor expression

The aim of the present study was to determine the effect of implanted neural stem cells (NSCs) on the functional recovery of tree shrews (TSs) subjected to hemi-sectioned spinal cord injury (hSCI), and to investigate the possible mechanism involved. NSCs (passage 2), derived from the hippocampus of TSs (embryonic day 20), were labeled with Hoechst 33342 and transplanted intraspinally into the hSC of TSs at thoracic level 10 in the acute (immediately after injury) and chronic (day 9 post-injury) stages. The Basso-Beattie-Bresnahan (BBB) score was recorded from days 1 to 16 post-injury, and the survival, migration, differentiation and neurotrophic factor (NTF) expression in vivo were detected. In vitro and in vivo, the expanded NSCs were able to differentiate into neurons and astrocytes, and secreted a variety of NTFs, including ciliary NTF, transforming growth factor-β1, glial cell line-derived NTF, nerve growth factor (NGF), brain-derived NTF and insulin-like growth factor. Following transplantation, the BBB score in the TSs with chronic-stage transplantation exhibited a statistically significant increase, while there was no significant difference in the acute group, compared with the control group. This corresponded with the marked upregulation of NGF indicated by reverse transcription-quantitative polymerase chain reaction. In conclusion, the transplantation of NSCs into the hSC in the chronic phase, but not the acute stage, of hSCI in non-human primate TSs is effective and associated with upregulated NGF expression. These findings may provide novel strategies for the treatment of SCI in clinical patients.

Erratum to: LPS Pretreatment Provides Neuroprotective Roles in Rats with Subarachnoid Hemorrhage by Downregulating MMP9 and Caspase3 Associated with TLR4 Signaling Activation

Subarachnoid hemorrhage (SAH), as a severe brain disease, has high morbidity and mortality. SAH usually induced neurological dysfunction or death and the treatment is far from satisfaction. Here, we investigated the effect of low dose of LPS pretreatment and underlying molecular mechanism in rat SAH model. Firstly, SAH model was induced by prechiasmal cistern injection method (SAH1) and common carotid artery-prechiasmal cistern shunt method (SAH2), respectively, to select the more suitable SAH model. At 6, 12, 24, 48, and 72 h after SAH, brain injury including neurological dysfunction, blood–brain barrier disruption, brain edema, and cell apoptosis were detected. And the expression of MMP9, HMGB1/TLR4, and caspase3 in cortex were also explored. Then, SB-3CT, an inhibitor of MMP9, was administrated to investigate the exact function of MMP9 in the brain injury at 24 h after SAH. Moreover, low dose of LPS was used to verify whether it had nerve protection after SAH and the mechanism involving in MMP9 and caspase 3 was investigated. Our results showed SAH1 seems to be the most suitable SAH model. In addition, MMP9 activated by HMGB1/TLR4 may promote or aggravate brain injury, while inhibiting MMP9 via SB-3CT exerted a neuroprotective effect. Moreover, LPS improved the neurological dysfunction, reduced Evans blue extravasation and brain edema, and inhibited cell apoptosis of cortex in rats with brain injury induced by SAH. Importantly, LPS pretreatment increased the expression level of TLR4, and decreased the level of MMP9 and caspase3. Therefore, the present study revealed that low dose of LPS pretreatment could provide neuroprotective effects on brain injury caused by SAH via downregulating MMP9 and caspase3 and activating TLR4 signal pathway.

Microarray expression profiles of genes in lung tissues of rats subjected to focal cerebral ischemia-induced lung injury following bone marrow-derived mesenchymal stem cell transplantation

Ischemia-induced stroke is the most common disease of the nervous system and is associated with a high mortality rate worldwide. Cerebral ischemia may lead to remote organ dysfunction, particular in the lungs, resulting in lung injury. Nowadays, bone marrow-derived mesenchymal stem cells (BMSCs) are widely studied in clinical trials as they may provide an effective solution to the treatment of neurological and cardiac diseases; however, the underlying molecular mechanisms remain unknown. In this study, a model of permanent focal cerebral ischemia-induced lung injury was successfully established and confirmed by neurological evaluation and lung injury scores. We demonstrated that the transplantation of BMSCs (passage 3) via the tail vein into the lung tissues attenuated lung injury. In order to elucidate the underlying molecular mechanisms, we analyzed the gene expression profiles in lung tissues from the rats with focal cerebral ischemia and transplanted with BMSCs using a Gene microarray. Moreover, the Gene Ontology database was employed to determine gene function. We found that the phosphoinositide 3-kinase (PI3K)-AKT signaling pathway, transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF) were downregulated in the BMSC transplantation groups, compared with the control group. These results suggested that BMSC transplantation may attenuate lung injury following focal cerebral ischemia and that this effect is associated with the downregulation of TGF-β, PDGF and the PI3K-AKT pathway.

Neural Stem Cell Transplantation Is Associated with Inhibition of Apoptosis, Bcl-xL Upregulation, and Recovery of Neurological Function in a Rat Model of Traumatic Brain Injury

Traumatic brain injury (TBI) is a common disease that usually causes severe neurological damage, and current treatment is far from satisfactory. The neuroprotective effects of neural stem cell (NSC) transplantation in the injured nervous system have largely been known, but the underlying mechanisms remain unclear, and their limited sources impede their clinical application. Here, we established a rat model of TBI by dropping a weight onto the cortical motor area of the brain and explored the effect of engrafted NSCs (passage 3, derived from the hippocampus of embryonic 12- to 14-d green fluorescent protein transgenic mice) on TBI rats. Moreover, RT-PCR and Western blotting were employed to investigate the possible mechanism associated with NSC grafts. We found rats with TBI exhibited a severe motor and equilibrium dysfunction, while NSC transplantation could partly improve the motor function and significantly reduce cell apoptosis and increase B-cell lymphoma–extra large (Bcl-xL) expression at 7 d postoperation. However, other genes including Bax, B-cell lymphoma 2, Fas ligand, and caspase3 did not exhibit significant differences in expression. Moreover, to test whether Bcl-xL could be used as a therapeutic target, herpes simplex virus (HSV) 1 carrying Bcl-xL recombinant was constructed and injected into the pericontusional cortices. Bcl-xL overexpression not only resulted in a significant improvement in neurological function but also inhibits cell apoptosis, as compared with the TBI rats, and exhibits the same effects as the administration of NSC. The present study therefore indicated that NSC transplantation could promote the recovery of TBI rats in a manner similar to that of Bcl-xL overexpression. Therefore, Bcl-xL overexpression, to some extent, could be considered as a useful strategy to replace NSC grafting in the treatment of TBI in future clinical practices.

Comparison of the properties of neural stem cells of the hippocampus in the tree shrew and rat in vitro

Neural stem cells (NSCs) are characterized by the ability of self-renewal and capacity to proliferate and produce new nervous tissue. NSCs are capable of differentiating to three lineages of neural cells, including neurons, oligodendrocytes and astrocytes. Furthermore, hippocampal NSCs transplantation can improve the neurological deficits associated with expression of cytokines. Therefore, to compare the properties of NSCs of tree shrews and rats in vitro, NSCs from tree shrews (tsNSCs) and rats f(rNSCs) were isolated. Nestin was used as a marker to identify the cultured NSCs. Neuronal nuclei protein and glial fibrillary acidic protein (GFAP) were utilized to demonstrate the differentiation of NSCs towards neurons and astrocytes, respectively, in vitro. Furthermore, the expression of neurotrophin 3 (NT3), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF) and transforming growth factor (TGF)β1 was also investigated in tsNSCs and rNSCs. The expression of all of the aforementioned proteins was detected using immunofluorescence methods. The results demonstrated that, after 5 days of culture, the average number of neurospheres in the cultured tsNSCs was significantly lower compared with rNSCs (P=0.0031). Additionally, compared with the rNSCs, tsNSCs exhibited an enhanced differentiation ability towards neurons. Furthermore, the expression of NT3 in the tsNSCs was higher compared with rNSCs (P<0.01), while the expression of BDNF was lower (P=0.045). However, no significant differences were observed in the expression level of GDNF and TGFβ1 between rNSCs and tsNSCs. Therefore, these results indicate that tsNSCs exhibit specific characteristics that are different from rNSCs, which provides novel information for the understanding of NSCs obtained from tree shrews. Overall, the results of the current study provide evidence to support the increased application of tree shrews as models for diseases of the central nervous system.

Knockdown of TNF‑α alleviates acute lung injury in rats with intestinal ischemia and reperfusion injury by upregulating IL‑10 expression

Intestinal ischemia and reperfusion (II/R) injury often triggers severe injury in remote organs, with the lungs being considered the main target. Excessive elevation of proinflammatory cytokines is a major contributor in the occurrence and development of II/R-induced acute lung injury (ALI). Therefore, the present study aimed to investigate whether blocking tumor necrosis factor-α (TNF-α) expression could protect the lungs from injury following II/R, and to explore the possible underlying mechanism involving interleukin-10 (IL-10). Briefly, II/R was induced in rats by 40 min occlusion of the superior mesenteric artery and celiac artery, followed by 8, 16 or 24 h of reperfusion. Subsequently, lentiviral vectors containing TNF-α short hairpin (sh)RNA were injected into the right lung tissues, in order to induce TNF-α knockdown. The severity of ALI was determined according to lung injury scores and lung edema (lung wet/dry weight ratio). The expression levels of TNF-α were analyzed by quantitative polymerase chain reaction (qPCR), western blotting and immunofluorescence (IF) staining. IL-10 expression, in response to TNF-α knockdown, was detected in lung tissues by qPCR and IF. The results detected marked inflammatory responses, and increased levels of lung wet/dry weight ratio and TNF-α expression, in the lungs of II/R rats. Conversely, treatment with TNF-α shRNA significantly alleviated the severity of ALI and upregulated the expression levels of IL-10 in lung tissues. These findings suggested that TNF-α RNA interference may exert a protective effect on II/R-induced ALI via the upregulation of IL-10. Therefore, TNF-α knockdown may be considered a potential strategy for the prevention or treatment of ALI induced by II/R in future clinical trials.