Synergic Neuroprotection Between Ligusticum Chuanxiong Hort and Borneol Against Ischemic Stroke by Neurogenesis via Modulating Reactive Astrogliosis and Maintaining the Blood–Brain Barrier

Background:Ligusticum chuanxiong Hort (LCH) is a famous ethnomedicine in Asia known for its excellent output on stroke treatment, and borneol usually acts as an assistant for its reducing permeability of the blood–brain barrier (BBB) after stroke. Although their synergy against brain ischemia was verified in previous studies, the potential mechanism is still unknown.Methods: The research aimed to explore the exact synergic mechanisms between LCH and borneol on neurogenesis within the areas of the dentate gyrus and subventricular zone. After treating middle cerebral artery occlusion rats with LCH (0.1 g/kg) and/or borneol (0.08 g/kg), the neurological severity score, brain infarct ratio, Nissl staining, Evans blue permeability, BBB ultrastructure, and expressions of von Willebrand factor and tight junction–associated proteins were measured. Co-localizations of Nestin+/BrdU+ and doublecortin+/BrdU+, and expressions of neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) were observed under a fluorescence microscope. Moreover, astrocyte polarization markers of complement component 3 and pentraxin 3, and relevant neurotrophins were also detected by immunoblotting.Results: Basically, LCH and borneol had different focuses, although both of them decreased infarct areas, and increased quantity of Nissl bodies and expression of brain-derived neurotrophic factor. LCH increased the neurological severity score, NeuN+ cells, and the ratios of Nestin+/BrdU+ and doublecortin+/BrdU+, and decreased GFAP+ cells and ciliary neurotrophic factor expression. Additionally, it regulated the expressions of complement component 3 and pentraxin 3 to transform astrocyte phenotypes. Borneol improved BBB ultrastructure and increased the expressions of von Willebrand factor, tight junction–associated proteins, vascular endothelial growth factor, and vascular endothelial growth factor receptor 2. Unexpectedly, their combined therapy showed more obvious regulations on the Nissl score, Evans blue permeability, doublecortin+/BrdU+, NeuN+ cells, brain-derived neurotrophic factor, and vascular endothelial growth factor than both of their monotherapies.Conclusions: The results indicated that LCH and borneol were complementary to each other in attenuating brain ischemia by and large. LCH mainly promoted neural stem cell proliferation, neurogenesis, and mature neuron preservation, which was probably related to the transformation of reactive astrocytes from A1 subtype to A2, while borneol preferred to maintain the integrity of the BBB, which provided neurogenesis with a homeostatic environment.

Rehabilitation Effects of Fatigue-Controlled Treadmill Training After Stroke: A Rat Model Study

Background: Traditional rehabilitation with uniformed intensity would ignore individual tolerance and introduce the second injury to stroke survivors due to overloaded training. However, effective control of the training intensity of different stroke survivors is still lacking. The purpose of the study was to investigate the rehabilitative effects of electromyography (EMG)-based fatigue-controlled treadmill training on rat stroke model.Methods: Sprague–Dawley rats after intracerebral hemorrhage and EMG electrode implantation surgeries were randomly distributed into three groups: the control group (CTRL, n = 11), forced training group (FOR-T, n = 11), and fatigue-controlled training group (FAT-C, n = 11). The rehabilitation interventions were delivered every day from day 2 to day 14 post-stroke. No training was delivered to the CTRL group. The rats in the FOR-T group were forced to run on the treadmill without rest. The fatigue level was monitored in the FAT-C group through the drop rate of EMG mean power frequency, and rest was applied to the rats when the fatigue level exceeded the moderate fatigue threshold. The speed and accumulated running duration were comparable in the FAT-C and the FOR-T groups. Daily evaluation of the motor functions was performed using the modified Neurological Severity Score. Running symmetry was investigated by the symmetry index of EMG bursts collected from both hind limbs during training. The expression level of neurofilament-light in the striatum was measured to evaluate the neuroplasticity.Results: The FAT-C group showed significantly lower modified Neurological Severity Score compared with the FOR-T (P ≤ 0.003) and CTRL (P ≤ 0.003) groups. The FAT-C group showed a significant increase in the symmetry of hind limbs since day 7 (P = 0.000), whereas the FOR-T group did not (P = 0.349). The FAT-C group showed a higher concentration of neurofilament-light compared to the CTRL group (P = 0.005) in the unaffected striatum and the FOR-T group (P = 0.021) in the affected striatum.Conclusion: The treadmill training with moderate fatigue level controlled was more effective in motor restoration than forced training. The fatigue-controlled physical training also demonstrated positive effects in the striatum neuroplasticity. This study indicated that protocol with individual fatigue-controlled training should be considered in both animal and clinical studies for better stroke rehabilitation.

Phospholipase D1 and D2 Synergistically Regulate Thrombus Formation

Previously, we reported that phospholipase D1 (PLD1) and PLD2 inhibition by selective PLD1 and PLD2 inhibitors could prevent platelet aggregation in humans, but not in mice. Moreover, only the PLD1 inhibitor, but not PLD2 inhibitor, could effectively prevent thrombus formation in mice, indicating that PLD might play different roles in platelet function in humans and mice. Although PLD1 and PLD2 were reported to be implicated in thrombotic events, the role of PLD in mice remains not completely clear. Here, we investigated the role of PLD1 and PLD2 in acute pulmonary thrombosis and transient middle cerebral artery occlusion-induced brain injury in mice. The data revealed that inhibition of PLD1, but not of PLD2, could partially prevent pulmonary thrombosis-induced death. Moreover, concurrent PLD1 and PLD2 inhibition could considerably increase survival rate. Likewise, inhibition of PLD1, but not PLD2, partially improved ischemic stroke and concurrent inhibition of PLD1, and PLD2 exhibited a relatively better protection against ischemic stroke, as evidenced by the infarct size, brain edema, modified neurological severity score, rotarod test, and the open field test. In conclusion, PLD1 might play a more important role than PLD2, and both PLD1 and PLD2 could act synergistically or have partially redundant functions in regulating thrombosis-relevant events.

Inhibition of microRNA-155 Alleviates Neurological Dysfunction Following Transient Global Ischemia and Contribution of Neuroinflammation and Oxidative Stress in the Hippocampus

Background/Aims: Central pro-inflammatory cytokine (PIC) signal is involved in neurological deficits after transient global ischemia induced by cardiac arrest (CA). The present study was to examine the role of microRNA- 155 (miR-155) in regulating IL-1β, IL-6 and TNF-α in the hippocampus of rats with induction of CA. We further examined the levels of products of oxidative stress 8-isoprostaglandin F2α (8-iso PGF2α, indication of oxidative stress); and 8-hydroxy-2’-deoxyguanosine (8-OHdG, indication of protein oxidation) after cerebral inhibition of miR-155. Methods: CA was induced by asphyxia and followed by cardiopulmonary resuscitation in rats. ELISA and western blot analysis were used to determine the levels of PICs and products of oxidative stress; and the protein expression of NADPH oxidase (NOXs) in the hippocampus. In addition, neurological severity score and brain edema were examined to assess neurological functions. Results: We observed amplification of IL-1β, IL-6 and TNF-α along with 8-iso PGF2α and 8-OHdG in the hippocampus of CA rats. Cerebral administration of miR-155 inhibitor diminished upregulation of PICs in the hippocampus. This also attenuated products of oxidative stress and upregulation of NOX4. Notably, inhibition of miR-155 improved neurological severity score and brain edema and this was linked to signal pathways of PIC and oxidative stress. Conclusion: We showed the significant role of blocking miR-155 signal in improving the neurological function in CA rats likely via inhibition of signal pathways of neuroinflammation and oxidative stress, suggesting that miR-155 may be a target in preventing and/or alleviating development of the impaired neurological functions during CA-evoked global cerebral ischemia.

The Effects of a Combination of Ion Channel Inhibitors in Female Rats Following Repeated Mild Traumatic Brain Injury

Following mild traumatic brain injury (mTBI), the ionic homeostasis of the central nervous system (CNS) becomes imbalanced. Excess Ca2+ influx into cells triggers molecular cascades, which result in detrimental effects. The authors assessed the effects of a combination of ion channel inhibitors (ICI) following repeated mTBI (rmTBI). Adult female rats were subjected to two rmTBI weight-drop injuries 24 h apart, sham procedures (sham), or no procedures (normal). Lomerizine, which inhibits voltage-gated calcium channels, was administered orally twice daily, whereas YM872 and Brilliant Blue G, inhibiting α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and P2X7 receptors, respectively, were delivered intraperitoneally every 48 h post-injury. Vehicle treatment controls were included for rmTBI, sham, and normal groups. At 11 days following rmTBI, there was a significant increase in the time taken to cross the 3 cm beam, as a sub-analysis of neurological severity score (NSS) assessments, compared with the normal control (p < 0.05), and a significant decrease in learning-associated improvement in rmTBI in Morris water maze (MWM) trials relative to the sham (p < 0.05). ICI-treated rmTBI animals were not different to sham, normal controls, or rmTBI treated with vehicle in all neurological severity score and Morris water maze assessments (p > 0.05). rmTBI resulted in increases in microglial cell density, antioxidant responses (manganese-dependent superoxide dismutase (MnSOD) immunoreactivity), and alterations to node of Ranvier structure. ICI treatment decreased microglial density, MnSOD immunoreactivity, and abnormalities of the node of Ranvier compared with vehicle controls (p < 0.01). The authors’ findings demonstrate the beneficial effects of the combinatorial ICI treatment on day 11 post-rmTBI, suggesting an attractive therapeutic strategy against the damage induced by excess Ca2+ following rmTBI.

TRPC1 Deficiency Exacerbates Cerebral Ischemia/Reperfusion-Induced Neurological Injury by Potentiating Nox4-Derived Reactive Oxygen Species Generation

Background/Aims: Transient receptor potential cation channel 1 (TRPC1)-mediated the calcium (Ca2+) influx plays an important role in several brain disorders. However, the function of TRPC1 in ischemia/reperfusion (I/R)-induced neurological injury is unclear. Methods: Wild-type or TRPC1 knockout mice underwent middle cerebral artery occlusion for 90 min followed by 24 h of reperfusion. In an in vitro study, neuronal cells were treated with oxygen–glucose deprivation and reoxygenation (OGD/R) to mimic I/R. The intracellular Ca2+ concentration [Ca2+]i was measured by Fura 2-AM under a microscope. Cerebral infarct volume was measured by triphenyltetrazolium chloride staining. Neurological function was examined by neurological severity score, Morris water maze test, rotarod test and string test. Oxidative parameters were detected by malondialdehyde, glutathione peroxidase, and superoxide dismutase commercially available kits. The protein expression levels of TRPC1, Nox4, p22phox, p47phox, and p67phox were analyzed by western blotting. Results: Brain tissues from cerebral I/R mice showed decreased TRPC1 expression. Similarly, TRPC1 expression was reduced in HT22 cells upon exposure to OGD/R treatment, followed by decreased Ca2+ influx. However, TRPC1 overexpression reversed the OGD/R-induced decrease in [Ca2+]i. TRPC1 knockout significantly exacerbated I/R-induced brain infarction, edema, neurological severity score, memory impairment, neurological deficits, and oxidative stress. In contrast, TRPC1 upregulation inhibited the increase in reactive oxygen species (ROS) generation induced by OGD/R. Analysis of key subunits of the Nox family and mitochondrial ROS revealed that the effects of TRPC1 downregulation on oxidative stress were associated with activation of Nox4-containing NADPH oxidase. TRPC1 interacted with Nox4 and facilitated Nox4 protein degradation under OGD/R conditions. In addition, TRPC1 inhibition potentiated the OGD/R-induced translocation of p47phox and p67phox as well as the interaction between Nox4 and p47phox or p67phox, whereas TRPC1 overexpression had the opposite effects. Conclusion: TRPC1 deficiency potentiates ROS generation via Nox4-containing NADPH oxidase, which exacerbates cerebral I/R injury. TRPC1 may be a promising molecular target for the treatment of stroke.

Distribution and Clearance of Blood Constituents after Intracerebral Hemorrhage in Rats: Mechanisms and Relevance for Functional Outcomes

Background and Objective To evaluate the impact of blood clearance mechanisms on neurologic damage and functional outcomes after intracerebral hemorrhage (ICH) in rats. Methods A rat model of ICH was established through pre-cannulation and injection of autologous arterial blood into the caudate nucleus. Blood was supplemented with 6-amino-caproic acid (a procoagulant), low-molecular-weight heparin (an anticoagulant), or vehicle. Relationships between hematoma volume and edema size and neurologic damage were assessed at different times. Results At 6 hours and 24 hours, hematoma volume was greater in rats that received anticoagulant than in the other two groups (p < 0.01). No significant differences were observed at 3 days, 5 days, or 7 days (p > 0.05). At 6 hours and 24 hours, the Neurological Severity Score in the procoagulant group was significantly higher than in the other two groups (comparison between groups, p < 0.01); and no significant differences were found at other times (p > 0.05). Conclusion Enhanced clearance of leaked blood was associated with larger hematomas within the first 3 days of ICH and with less neurologic damage. The capacity to clear blood is an important determinant of functional recovery after ICH. Enhanced clearance may help reduce neurologic damage.

GSK-3β Inhibition Induced Neuroprotection, Regeneration, and Functional Recovery after Intracerebral Hemorrhagic Stroke

Hemorrhagic stroke is a devastating disease that lacks effective therapies. In the present investigation, we tested 6-bromoindirubin-3′-oxime (BIO) as a selective glycogen synthase kinase-3β (GSK-3β) inhibitor in a mouse model of intracerebral hemorrhage (ICH). ICH was induced by injection of collagenase IV into the striatum of 8- to 10-week-old C57BL/6 mice. BIO (8 μg/kg, IP) was administered following either an acute delivery (0–2 h delay) or a prolonged regimen (every 48 h starting at 3 days post-ICH). At 2 days post-ICH, the acute BIO treatment significantly reduced the hematoma volume. In the perihematoma regions, BIO administration blocked GSK-3β phosphorylation/activation, increased Bcl-2 and β-catenin levels, and significantly increased viability of neurons and other cell types. The prolonged BIO regimen maintained a higher level of β-catenin, upregulated VEGF and BDNF, and promoted neurogenesis and angiogenesis in peri-injury zones at 14 days after ICH. The BIO treatment also promoted proliferation of neural stem cells (NSCs) and migration of nascent DCX+ neuroblasts from the subventricular zone (SVZ) to the lesioned cortex. BIO improved functional outcomes on both the neurological severity score and rotarod tests. The findings of this study corroborate the neuroprotective and regenerative effects of BIO and suggest that the Wnt/GSK-3β/β-catenin pathway may be explored for the treatment of acute or chronic ICH.

Neuronal Regeneration after Electroacupuncture Treatment in Ischemia–Reperfusion-Injured Cerebral Infarction Rats

Adult neuronal cells which can regenerate have been reported. The present study investigated whether acupuncture enhances neuronal regeneration in ischemic stroke rats. We established an ischemic stroke rat model by occluding the cerebral blood flow of the right middle cerebral artery for 15 minutes and then allowing reperfusion in Sprague–Dawley rats. The results indicated that, in these rats, 2 Hz electroacupuncture (EA) at both Zusanli (ST36) and Shangjuxu (ST37) acupoints reduced the infarction/hemisphere ratio 8 days after reperfusion and reduced the modified neurological severity score (mNSS) and increased the rotarod test time 4 and 8 days after reperfusion, respectively. In addition, 2 Hz reduced nestin immunoreactive cells in the penumbra area and the ischemic core area; 2 Hz EA also reduced Ki67 immunoreactive cells and increased glial fibrillary acidic protein immunoreactive cells in the penumbra area. These findings suggest that 2 Hz EA at the ST36 and ST37 acupoints has a neuroprotective role. However, additional studies are needed to further investigate these preliminary results.

Abstract WP298: Matrix Metalloproteinases Are Involved In The Regulation Of Angiogenesis After Intracerebral Hemorrhage

Background and purpose: Intracerebral hemorrhage (ICH) is one of the most devastating subtypes of stroke. And our previous work has demonstrated that ICH induces angiogenesis, accompanied by up-regulation of pro-angiogenic factors. Matrix metalloproteinases (MMPs) can cause blood-brain barrier dysfunction by degrading the extracellular cellular matrix (ECM) around the vessels after ICH, but opening a way for the prolonging newborn vessels is a key step for their functional structure, therefore, the purpose of the study is to investigate whether MMPs are involved in the process of angiogenesis after ICH. Methods: Thirty Kunming mice were randomly divided into sham group, ICH group and doxycycline (DOX)-treated group. And then 5 mice were randomly selected for Western Blot to detect the expression of MMP9, and the other five for the immunohistochemistry to detect vWF. ICH model was induced by injection collagenase type VII into right globus pallidus stereotaxically, and DOX, a broad-spectrum MMP inhibitor, was injected by intraperitoneally at 7 days after ICH induction. Neurological severity score (NSS), corner turn test and foot-fault test were used to investigate the neurological function. And vWF-positive vessels were counted around the hematoma. Results: At 7 days, there is no difference between the two ICH-induced groups in NSS, corner turn test, foot-fault test; while at 14 days, the NSS in ICH group is significantly lower than that of DOX-treated group ( P <0.05), and the times for right-turn and foot-fault in ICH group are notably fewer than those of DOX-treated group ( P <0.05); At 14d, the number of vWF-postive microvessel in ICH group was significantly larger than that of DOX-treated group ( P <0.01), and Western Blot revealed that DOX decreased MMP9 expression remarkably( P <0.01). Conclusion: Matrix metalloproteinases were involved in the regulation of angiogenesis after ICH.