Ming Fu Chiang

Enhancement of neuroplasticity through upregulation of β1-integrin in human umbilical cord-derived stromal cell implanted stroke model

Neuroplasticity subsequent to functional angiogenesis is an important goal for cell-based therapy of ischemic neural tissues. At present, the cellular and molecular mechanisms involved are still not well understood. In this study, we isolated mesenchymal stem cells (MSCs) from Whartons jelly (WJ) to obtain clonally expanded human umbilical cord-derived mesenchymal stem cells (HUCMSCs) with multilineage differentiation potential. Experimental rats receiving intracerebral HUCMSC transplantation showed significantly improved neurological function compared to vehicle-treated control rats. Cortical neuronal activity, as evaluated by proton MR spectroscopy (1H-MRS), also increased considerably in the transplantation group. Transplanted HUCMSCs migrated towards the ischemic boundary zone and differentiated into glial, neuronal, doublecortin+, CXCR4+, and vascular endothelial cells to enhance neuroplasticity in the ischemic brain. In addition, HUCMSC transplantation promoted the formation of new vessels to increase local cortical blood flow in the ischemic hemisphere. Modulation by stem cell-derived macrophage/microglial interactions, and increased beta1-integrin expression, might enhance this angiogenic architecture within the ischemic brain. Inhibition of beta1-integrin expression blocked local angiogenesis and reduced recovery from neurological deficit. In addition, significantly increased modulation of neurotrophic factor expression was also found in the HUCMSC transplantation group. In summary, regulation of beta1-integrin expression plays a critical role in the plasticity of the ischemic brain after the implantation of HUCMSCs.

Intracerebral peripheral blood stem cell (CD34+) implantation induces neuroplasticity by enhancing β1 integrin-mediated angiogenesis in chronic stroke rats

Although stem cell-based treatments for stroke and other neurodegenerative diseases have advanced rapidly, there are still few clinical treatments available. In this study, rats receiving intracerebral peripheral blood hematopoietic stem cell (CD34+) (PBSC) transplantation showed much more improvement in neurological function after chronic cerebral ischemia in comparison with vehicle-treated control rats. Using laser-scanning confocal microscopy, implanted PBSCs were seen to differentiate into glial cells [GFAP+ (glial fibrillary acidic protein-positive)], neurons [Nestin+, MAP-2+ (microtubule-associated protein 2-positive), Neu-N+ (neuronal nuclear antigen-positive)], and vascular endothelial cells [vWF+ (von Willebrand factor-positive)], thereby enhancing neuroplastic effects in the ischemic brain. Cortical neuronal activity, as evaluated by 1H-MRS (proton magnetic resonance spectroscopy), also increased considerably in PBSC-treated rats compared with a vehicle-treated control group. In addition, PBSC implantation promoted the formation of new vessels, thereby increasing the local cortical blood flow in the ischemic hemisphere. These observations may be explained by the involvement of stem cell-derived macrophage/microglial cells, and β1 integrin expression, which might enhance this angiogenic architecture over the ischemic brain. Furthermore, quantitative reverse transcription-PCR analysis showed significantly increased modulation of neurotrophic factor expression in the ischemic hemisphere of the PBSC-transplanted rats compared with vehicle-treated control rats. Thus, intracerebral PBSC transplantation might have potential as a therapeutic strategy for treating cerebrovascular diseases.

Overexpression of PrPC by adenovirus-mediated gene targeting reduces ischemic injury in a stroke rat model

Prion diseases are induced by pathologically misfolded prion protein (PrPSc), which recruit normal sialoglycoprotein PrPC by a template-directed process. In this study, we investigated the expression of PrPC in a rat model of cerebral ischemia to more fully understand its physiological role. Immunohistochemical analysis demonstrated that PrPC-immunoreactive cells increased significantly in the penumbra of ischemic rat brain compared with the untreated brain. Western blot analysis showed that PrPC protein expression increased in ischemic brain tissue in a time-dependent manner. In addition, PrPC protein expression was seen to colocalize with neuron, glial, and vascular endothelial cells in the penumbric region of the ischemic brain. Overexpression of PrPC by injection of rAd (replication-defective recombinant adenoviral)-PGK (phosphoglycerate kinase)-PrPC-Flag into ischemic rat brain improved neurological behavior and reduced the volume of cerebral infarction, which is supportive of a role for PrPC in the neuroprotective adaptive cellular response to ischemic lesions. Concomitant upregulation of PrPC and activated extracellular signal-regulated kinase (ERK1/2) under hypoxia–reoxygenation in primary cortical cultures was shown to be dependent on ERK1/2 phosphorylation. During hypoxia–reoxygenation, mouse neuroblastoma cell line N18 cells transfected with luciferase rat PrPC promoter reporter constructs, containing the heat shock element (HSE), expressed higher luciferase activities (3- to 10-fold) than those cells transfected with constructs not containing HSE. We propose that HSTF-1 (hypoxia-activated transcription factor), phosphorylated by ERK1/2, may in turn interact with HSE in the promoter of PrPC resulting in gene expression of the prion gene. In summary, we conclude that upregulation of PrPC expression after cerebral ischemia and hypoxia exerts a neuroprotective effect on injured neural tissue. This study suggests that PrPC has physiological relevance to cerebral ischemic injury and could be useful as a therapeutic target for the treatment of cerebral ischemia.

Secretoneurin promotes neuroprotection and neuronal plasticity via the Jak2/Stat3 pathway in murine models of stroke

Secretoneurin (SN), a neuropeptide derived from secretogranin II, promotes neurite outgrowth of immature cerebellar granule cells. SN also aids in the growth and repair of neuronal tissue, although the precise mechanisms underlying the promotion of brain tissue neuroprotection and plasticity by SN are not understood. Here, in a rat model of stroke and in ischemic human brain tissue, SN was markedly upregulated in both neurons and endothelial cells. SN-mediated neuroprotection rescued primary cortical cell cultures from oxygen/glucose deprivation. SN also induced expression of the antiapoptotic proteins Bcl-2 and Bcl-xL through the Jak2/Stat3 pathway and inhibited apoptosis by blocking caspase-3 activation. In addition, rats with occluded right middle cerebral arteries showed less cerebral infarction, improved motor performance, and increased brain metabolic activity following i.v. administration of SN. Furthermore, SN injection enhanced stem cell targeting to the injured brain in mice and promoted the formation of new blood vessels to increase local cortical blood flow in the ischemic hemisphere. Both in vitro and in vivo, SN not only promoted neuroprotection, but also enhanced neurogenesis and angiogenesis. Our results demonstrate that SN acts directly on neurons after hypoxia and ischemic insult to further their survival by activating the Jak2/Stat3 pathway.

Association between apolipoprotein E genotype and outcome of traumatic brain injury

Summary¶Background. The prognosis of traumatic brain injury is quite variable and not fully explained by the known factors. This study is to examine if the polymorphism of apolipoprotein E (apoE) influences the outcome of traumatic brain injury.Methods. Over a period of twelve months, we prospectively studied 100 patients who sustained traumatic brain injuries and were admitted to our neurosurgical unit.Findings. Nineteen patients were apoE4(+) and 81 patients were apoE4(−). There was no significant difference between apoE4(+) and apoE4(−) groups in the cause of injury (p=0.288), type of brain injury (p=0.983) and choice of treatment (p=0.88). The proportion of patients with a lower GCS (<13), indicating a poor prognosis, was higher in the apoE4(+) group (73.7%) than that in apoE4(−) group (61.7%), although the difference was not significant (p=0.654). Six patients (7.4%) in the apoE4(−) group and 5(26.3%) in the apoE4(+) group had been drinking alcohol at the time of injury (p=0.018). The mean duration of hospital stay for apoE4(+) patients was significantly longer than for apoE4(−) patients (p<0.001). Six months after injury, 10 of 19 patients (52.6%) with apoE4 had an unfavorable outcome (dead, vegetative state, or severe disability) compared with 20 of the 81 (24.1%) patients without apoE4 (p=0.017). The apoE4(+) patients had a significantly longer hospital stay and unfavorable outcomes after brain injury.Interpretation. This study discloses a significant genetic association between the apoE genotypes and outcomes of traumatic brain injury. Patients with apoE4 allele are more likely to have an unfavorable clinical outcome after brain injury.

Neuregulin-1 reduces ischemia-induced brain damage in rats

Neuregulin-1 (NRG-1) is expressed throughout the immature and adult central nervous system and it has been demonstrated to influence the migration of a variety of cell types in developing brain. Elevated levels of NRG-1 transcript are found in the adult brain after injury, leading to the suggestion that NRG-1 is involved in the physiological response to neuronal injury. Here, we report our findings that rats pre-treated with NRG-1 protein, undergoing cerebral ischemia 30 min later, had increased motor performance and less cerebral infarction than untreated rats. In the cortex of NRG-1 treated rats, ischemia induced a decrease in caspase-3 immunoreactivity and a reduction in the density of cells positive for terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end-labeling. Improvement in behavioral assays was also found in animals treated with NRG-1. Pre-treatment with NRG-1 did not alter cerebral blood flow or other physiological parameters. NRG-1 reduced ischemia/reperfusion injury, indicating that it may act as an endogenous neuroprotective factor against stroke. Therefore, NRG-1 may represent a target for the development of new treatments for stroke.

Hyperbaric oxygen enhances the expression of prion protein and heat shock protein 70 in a mouse neuroblastoma cell line

Abstract1. Cellular prion protein, PrPC, is a ubiquitous glycoprotein strongly expressed in neurons with an as yet unknown biological function. In previous studies, we demonstrated that PrPC could be regulated by heat shock stress, implying that it might be a stress-responsive protein. Hyperbaric oxygen (HBO) administration is a well-defined model for the study of oxidative stress.2. This study investigated the effect of HBO on PrPC and Hsp 70 expression in mouse neuroblastoma cell lines (N18), assessing the expression of PrPC and Hsp 70 using RT-PCR and Western blotting. HBO administration resulted in a time- and dose-dependent increase in PrPC and Hsp70 expression in N18 cells at both mRNA and protein levels, with a concomitant upregulation of c-Jun N-terminal kinase (JNK).3. Under HBO treatment, luciferase reporter constructs of the rat PrPC promoter, containing the heat shock element (HSE) also present in Hsp70, expressed higher luciferase activity (3- to 10-fold) than those constructs without HSE.4. In summary, these data suggest that PrPC and Hsp 70 may be regulated by HBO, through the activation of JNK. Thus, the activated heat shock transcriptional factor 1, phosphorylated by JNK interacted with HSE in the promoter of PrPC resulted in increased gene expression. These findings are vital for future therapeutic approaches in transmissible spongiform encephalopathies and the understanding of the function of the PrPC.

Hypoglycemia enhances the expression of prion protein and heat-shock protein 70 in a mouse neuroblastoma cell line

Cellular prion protein (PrPC) expression can be regulated by heat‐shock stress, and we designed the present study to determine whether hypoglycemia could affect PrPC expression. RT‐PCR and Western blotting were used to measure the expression of PrPC and heat‐shock protein (Hsp70) in mouse neuroblastoma (N18) cells cultured 3 hr to 3 days in media deprived of 97.5% (L) or 75% (M) of its glucose. Hypoglycemia caused a concomitant time‐dependent and glucose dose‐dependent increase in PrPC and Hsp70. In addition, hypoglycemia also increased phosphorylated c‐Jun N‐terminal kinase (JNK) protein levels in a time‐dependent manner. The upregulation of PrPC and Hsp70 under hypoglycemic conditions was disrupted by the specific JNK inhibitor SP600125. It was also found from in vitro studies that hypoglycemic conditions induced higher levels of PrPC promoter activity in PrPC promoters containing a heat‐shock element (HSE) than in PrPC promoters lacking HSE. We propose that hypoglycemia‐increased PrPC expression might be due to JNK phosphorylation of a heat‐shock transcriptional factor, which then interacts with HSE in the promoter of PrPC.

Brain Single Photon Emission Computed Tomography in Patients with A3243G Mutation in Mitochondrial DNA tRNA

Abstract: Brain single photon emission computed tomography (SPECT) studies were conducted in three patients with A3243G mutation of the mitochondrial (mt) DNA tRNA. All were born to mothers suffering from chronic progressive external ophthalmoplegia (CPEO) with the same A3243G point mutation of the mtDNA tRNA. The first case manifested clinically with MELAS, the second case manifested with CPEO, and third case was characterized by recurrent migraine‐like headache, tremor, and epilepsy. Brain SPECT of all patients, regardless of whether they had or had not suffered from stroke‐like episodes, showed multiple areas of asymmetrical decreased perfusion, particularly in the posterior and lateral head regions, especially the temporal lobes. Crossed‐cerebellar diaschisis may occur. Conventional brain magnetic resonance images failed to show some of the lesions. Decreased regional cerebral blood flow, rather than previously proposed hyperemia, is likely to be the cause. We conclude that mitochondrial vasculopathy with regional cerebral hypoperfusion may be seen on brain SPECT in patients with mitochondrial disorders and A3243G mutations, regardless of whether they have or have not suffered from stroke‐like episodes.

Multiparametric analysis of cerebral substrates and nitric oxide delivery in cerebrospinal fluid in patients with intracerebral haemorrhage: correlation with hemodynamics and outcome

SummaryBackground. There is no information regarding the possible role of cerebral substrates in the pathogenesis of neuronal injury in intracerebral haemorrhages (ICHs). Purposes of this prospective study were to clarify whether changes in substrates are the consequence of the initial brain damage in ICH and to elucidate the relationship among the biochemical mechanisms and clinical course of patients with ICH. Method. During a period of two years, patients (GCS ≤8) who had ICH secondary to an aneurysm (SAH), stroke (sICH), or trauma (tICH) and underwent ventriculostomy with ICP monitoring and/or underwent cranial surgery were randomly enrolled in this study. Extracellular concentrations of glutamate, aspartate, glycine, GABA, lactate, lactate/pyruvate ratio, and glucose in the CSF were measured by use of high-performance liquid chromatography (HPLC). The nitric oxide (NO) concentration in the CSF was analyzed by chemiluminescence. Findings. There were 75 patients (38 women and 37 men) with ICH included in this study. Twenty-one patients had SAH, 28 sICH, and 26 tICH. In tICH patients, there was a 30-fold increase in glutamate and a 10-fold in aspartate over reference values. The levels of glutamate, aspirate, GABA, lactate, glucose, and NO differed significantly among the three groups (p<0.001). There were no significant differences in glycine and L/P ratio among the groups. The initial GCS, the mean CPP and outcome six months after the insult were all significantly correlated with the concentration of substrates (p<0.01), both within groups and among the total sample. The CSF levels of glutamate lactate, NO and glucose correlated significantly with outcome (p<0.005). Conclusions. This study confirms the correlation between the level of EAAs and the outcome of ICHs, suggesting that neurochemical monitoring of these substances may have a role in caring for patients.