Neuroprotection by dimethyloxalylglycine following permanent and transient focal cerebral ischemia in rats

Dimethyloxalylglycine (DMOG) is an inhibitor of prolyl-4-hydroxylase domain (PHD) enzymes that regulate the stability of hypoxia-inducible factor (HIF). We investigated the effect of DMOG on the outcome after permanent and transient middle cerebral artery occlusion (p/tMCAO) in the rat. Before and after pMCAO, rats were treated with 40 mg/kg, 200 mg/kg DMOG, or vehicle, and with 40 mg/kg or vehicle after tMCAO. Serial magnetic resonance imaging (MRI) was performed to assess infarct evolution and regional cerebral blood flow (rCBF). Both doses significantly reduced infarct volumes, but only 40 mg/kg improved the behavior after 24 hours of pMCAO. Animals receiving 40 mg/kg were more likely to maintain rCBF values above 30% from the contralateral hemisphere within 24 hours of pMCAO. DMOG after tMCAO significantly reduced the infarct volumes and improved behavior at 24 hours and 8 days and also improved the rCBF after 24 hours. A consistent and significant upregulation of both mRNA and protein levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) was associated with the observed neuroprotection, although this was not consistently related to HIF-1α levels at 24 hours and 8 days. Thus, DMOG afforded neuroprotection both at 24 hours after pMCAO and at 24 hours and 8 days after tMCAO. This effect was associated with an increase of VEGF and eNOS and was mediated by improved rCBF after DMOG treatment.     

大鼠短暂局灶性大脑中动脉缺血后calpain的表达

目的：研究calpain在缺血性脑损伤中的作用，进一步探讨缺血性脑血管病的分子机制，为治疗研发提供理论依据。方法：用Belayev改良的Langa线栓法制备大鼠局灶性大脑中动脉(MCA)缺血／再灌注模型，TTC染色观察梗死灶的形成，分别用原位杂交及免疫组化技术检测鼠脑中calpain mRNA与活性蛋白的表达。结果：缺血2h再灌注24h,TTC染色见明显的梗死灶形成，正常脑组织、假手术组及：MCAO缺血对侧脑中有少量的calpain mRNA表达，但活性蛋白几无表达；缺血脑组织calpain mRNA表达及蛋白质活化均显著增加，呈双峰式，MCA缺血2h增加，再灌注4h减少，至24h更明显增高，而48h又有所下降。结论：Calpain参与了缺血性脑损伤过程，尤其在迟发性神经元死亡中起重要作用。     

Ziprasidone attenuates brain injury after focal cerebral ischemia induced by middle cerebral artery occlusion in rats

Ziprasidone is an atypical antipsychotic drug used for the treatment of schizophrenia. Recent studies have reported that atypical antipsychotics have neuroprotective effects against brain injury. In the present study, the effect of ziprasidone on ischemic brain injury was investigated. Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in rats. All the animals experienced ischemia for 1h and then underwent reperfusion. The infarct size induced by MCAO was significantly reduced in the animals that received acute treatment with 5mg/kg ziprasidone and subchronic treatment with 2.5mg/kg ziprasidone for 7 days compared with that in the vehicle-treated animals. The acute treatment with ziprasidone significantly improved neurological functions, as measured by the modified neurological severity score, in a dose-dependent manner. The subchronic treatment produced more rapid recovery from functional deficits than the vehicle treatment. The immunohistochemical investigation revealed that the subchronic treatment prevented severe loss of neuronal marker intensity and attenuated the increased in microglial marker intensity in the infarcted cortical area. These results suggest that ziprasidone has neuroprotective effects in a rat model of ischemic stroke and provide new insight for its clinical applications.     

Late changes in blood–brain barrier permeability in a rat tMCAO model of stroke detected by gadolinium-enhanced MRI

ABSTRACT

Objectives

After cerebral ischaemia the blood–brain barrier (BBB) may be compromised and this has been observed in both clinical and preclinical studies. The timing of BBB disruption after ischaemia has long been considered to be biphasic, however some groups contest this view. Therefore, the purpose of this study was to characterize the BBB permeability timecourse in a rat model at both acute and chronic time points     

Decomposition and long-lasting downregulation of extracellular matrix in perineuronal nets induced by focal cerebral ischemia in rats

The upregulation of extracellular matrix components, especially chondroitin sulfate proteoglycans, after brain injury and stroke is known to accompany the glial reaction, forming repellent scars that hinder axonal growth and the reorganization of the injured neuronal networks. The extracellular matrix associated with perineuronal nets (PNs) in the primarily injured and remote regions has not yet been systematically analyzed. We use the model of permanent middle cerebral artery occlusion (MCAO) to investigate the acute and long-lasting consequences of ischemia for PNs, related to the damage of neurons and reactions of glial cells, in spontaneously hypertensive rats. Extracellular matrix components associated with PNs around cortical interneurons and neurons in thalamic nuclei were characterized 1, 7, 14, and 35 days after MCAO, using Wisteria floribunda agglutinin (WFA) staining and immunocytochemistry. The degradation of PNs in the infarct core was initiated by loss of WFA-binding matrix components, indicating the cleavage of glycosaminoglycan chains of chondroitin sulfate proteoglycans. Immunostaining showed the subsequent removal of proteoglycan core proteins within the extending microglia/macrophage invasion zone lasting for 2 weeks after MCAO. In the cortical periinfarct region, delineated by an astrocytic scar against the infarct core, the number of WFA-stained and proteoglycan core protein-immunoreactive PNs was permanently reduced. In the homolateral ventroposterior thalamus, the delayed decrease in perineuronal matrix was related to the distribution pattern of activated microglia and massive neuronal degeneration. It can be concluded from these results that complementary to the known upregulation of matrix components in the glial scar, deficits in the expression of the neuron-associated extracellular matrix develop in the periinfarct and remote regions. These deficits may contribute to the long-lasting functional impairments after stroke.     

Lack of iNOS induction in a severe model of transient focal cerebral ischemia in rats

Calcium-independent nitric oxide synthase (NOS) activity has been reported in ischemic brains and usually attributed to the inducible isoform, iNOS. Because calcium-independent mechanisms have recently been shown to regulate the constitutive calcium-dependent NOS, we proposed to confirm the presence of iNOS activity in our model of transient focal cerebral ischemia in rats. Our initial results showed that, in our model, ischemia induced an important increase in brain calcium concentration. Consequently, the determination of calcium-independent NOS activity required a higher concentration of calcium chelator than classically used in the NOS assay. In these conditions, calcium-independent NOS activity was not observed after ischemia. Moreover, our ischemia was associated with neither iNOS protein expression, measured by Western blotting, nor increased NO production, evaluated by its metabolites (nitrate/nitrite). Our results demonstrate that iNOS activity may be overestimated due to increased brain calcium concentration in ischemic conditions and also that iNOS is not systematically induced after cerebral ischemia.     

Therapeutic imaging window of cerebral infarction revealed by multisequence magnetic resonance imaging An animal and clinical study

In this study, we established a Wistar rat model of right middle cerebral artery occlusion and observed pathological imaging changes (T2-weighted imaging [T2WI], T2FLAIR, and diffusion-weighted imaging [DWI]) following cerebral infarction. The pathological changes were divided into three phases: early cerebral infarction, middle cerebral infarction, and late cerebral infarction. In the early cerebral infarction phase (less than 2 hours post-infarction), there was evidence of intracellular edema, which improved after reperfusion. This improvement was defined as the ischemic penumbra. In this phase, a high DWI signal and a low apparent diffusion coefficient were observed in the right basal ganglia region. By contrast, there were no abnormal T2WI and T2FLAIR signals. For the middle cerebral infarction phase (2-4 hours post-infarction), a mixed edema was observed. After reperfusion, there was a mild improvement in cell edema, while the angioedema became more serious. A high DWI signal and a low apparent diffusion coefficient signal were observed, and some rats showed high T2WI and T2FLAIR signals. For the late cerebral infarction phase (4-6 hours post-infarction), significant angioedema was visible in the infarction site. After reperfusion, there was a significant increase in angioedema, while there was evidence of hemorrhage and necrosis. A mixed signal was observed on DWI, while a high apparent diffusion coefficient signal, a high T2WI signal, and a high T2FLAIR signal were also observed. All 86 cerebral infarction patients were subjected to T2WI, T2FLAIR, and DWI. MRI results of clinic data similar to the early infarction phase of animal experiments were found in 51 patients, for which 10 patients (10/51) had an onset time greater than 6 hours. A total of 35 patients had MRI results similar to the middle and late infarction phase of animal experiments, of which eight patients (8/35) had an onset time less than 6 hours. These data suggest that defining the "therapeutic time window" as the time 6 hours after infarction may not be suitable for all patients. Integrated application of MRI sequences including T2WI, T2FLAIR, DW-MRI, and apparent diffusion coefficient mapping should be used to examine the ischemic penumbra, which may provide valuable information for identifying the "therapeutic time window".     

Analysis of ischemic cerebral lesions using 3.0-T diffusion-weighted imaging and magnetic resonance angiography after revascularization surgery for ischemic disease

Background

Cerebral revascularization surgery (CRS) is increasingly recognized as an important component in the treatment of complex cerebral vascular disease and tumors. CRS requires that the incidence of perioperative neurological complications should be minimized, because CRS for ischemic disease is often not the goal of treatment, but rather a prophylactic surgery. CRS carries the risk of focal postoperative neurological deficits. Little has been established concerning mechanisms of post-CRS ischemia. We used 3.0-T diffusion-weighted magnetic resonance imaging (DWI) and magnetic resonance angiography (MRA) to analyze the incidence and mechanism of ischemic lesions.

Methods

We studied the anterior circulation territory after 20 CRS procedures involving 33 vascular anastomosis procedures (13 double anastomoses and 7 single anastomoses) in 12 men and 8 women between June 2007 and October 2011. The operations included single or double superficial temporal artery–middle cerebral artery (STA–MCA) anastomosis to treat internal carotid artery/MCA occlusions or severe MCA stenosis. A combined STA–MCA anastomosis and indirect bypass were performed for moyamoya disease. Postoperative DWI and MRA were obtained in all patients between 24 and 96 h after surgery to detect thromboembolism, hypoperfusion, or procedural ischemic complications and vasospasms of the donor STA.

Results

Follow-up DWI and MRA were carried out 1.8 ± 0.6 days after CRS (range, 1–4 days). Temporary occlusion time for anastomoses averaged 18.9 min (range, 16–32 min). Asymptomatic new hyperintensities occurred in the ipsilateral hemisphere of 2 patients on postoperative DWI (10% patients/6.0% anastomoses), and 1 moyamoya patient (5.0% patients/3.0% anastomoses) developed a symptomatic hyperintensity in the ipsilateral occipital lobe in response to the operation. Two abnormal small (<5 mm) cortical DWI lesions were caused by sacrifices of a small branch of the recipient MCA.

Conclusion

This study is the first postoperative 3.0-T DWI study of CRS and related clinical events. The incidence of symptomatic postoperative DWI abnormalities was restricted to 1 moyamoya patient representing 5.0% of total patients and 3.0% anastomoses. Although some postoperative DWI abnormalities occurred, CRS was found to be safe with a low risk of symptomatic ischemia.     

Characterization of astrocytes and microglial cells in the hippocampal CA1 region after transient focal cerebral ischemia in rats treated with Ilexonin A

Ilexonin A is a compound isolated from the root of Ilex pubescens,a traditional Chinese medicine.Ilexonin A has been shown to play a neuroprotective role by regulating the activation of astrocytes and microglia in the peri-infarct area after ischemia.However,the effects of ilexonin A on astrocytes and microglia in the infarct-free region of the hippocampal CA1 region remain unclear.Focal cerebral ischemia models were established by 2-hour occlusion of the middle cerebral artery in rats.Ilexonin A(20,40 or 80 mg/kg)was administered immediately after ischemia/reperfusion.The astrocyte marker glial fibrillary acidic protein,microglia marker Iba-1,neural stem cell marker nestin and inflammation markers were detected by immunohistochemistry and western blot assay.Expression levels of tumor necrosis factor-αand interleukin 1βwere determined by enzyme linked immunosorbent assay in the hippocampal CA1 tissue.Astrocytes were activated immediately in progressively increasing numbers from 1,3,to 7 days post-ischemia/reperfusion.The number of activated astrocytes further increased in the hippocampal CA1 region after treatment with ilexonin A.Microglial cells remained quiescent after ischemia/reperfusion,but became activated after treatment with ilexonin A.Ilexonin A enhanced nestin expression and reduced the expression of tumor necrosis factor-αand interleukin 1βin the hippocampus post-ischemia/reperfusion.The results of the present study suggest that ilexonin A has a neuroprotective effect in the hippocampus after ischemia/reperfusion,probably through regulating astrocytes and microglia activation,promoting neuronal stem cell proliferation and reducing the levels of pro-inflammatory factors.This study was approved by the Animal Ethics Committee of the Fujian Medical University Union Hospital,China.     

Blood-letting punctures at twelve Jing-Well points of the hand can treat cerebral ischemia in a similar manner to mannitol

A rat model of middle cerebral artery permanent occlusion was established using the modified Longa method. Successfully established model animals were treated by blood-letting puncture at twelve Jing-Well points of the hand, and/or by injecting mannitol into the caudal vein twice daily. Brain tissue was collected at 24, 48 and 72 hours after modeling, and blood was collected through the retinal vein before Evans blue was injected, approximately 1 hour prior to harvesting of brain tissue. Results showed that Evans blue leakage into brain tissue and serum nitric oxide synthase activity were significantly increased in model rats. Treatment with blood-letting punctures at twelve Jing-Well points of the hand and/or injection of mannitol into the caudal vein reduced the amount of Evans blue leakage into the brain tissue and serum nitric oxide synthase activity to varying degrees. There was no significant difference between single treatment and combined treatment. Experimental findings indicate that blood-letting punctures at twelve Jing-Well points of the hand can decrease blood-brain barrier permeability and serum nitric oxide synthase activity in rats following middle cerebral artery occlusion, and its effect is similar to that of mannitol injection alone and Jing-Well points plus mannitol injection.     

Protection by neuroglobin and cell-penetrating peptide-mediated delivery in vivo: a decade of research. Comment on Cai et al: TAT-mediated delivery of neuroglobin protects against focal cerebral ischemia in mice. Exp Neurol. 2011; 227(1): 224-31

Over the last decade, numerous studies have suggested that neuroglobin is able to protect against the effects of ischemia. However, such results have mostly been based on models using transgenic overexpression or viral delivery. As a therapy, new technology would need to be applied to enable delivery of high concentrations of neuroglobin shortly after the patient suffers the stroke. An approach to deliver proteins in ischemia in vivo in a timely manner is the use of cell-penetrating peptides (CPP). CPP have been used in animal models for brain diseases for about a decade as well. In a recent issue of Experimental Neurology, Cai and colleagues test the effect of CPP-coupled neuroglobin in an in vivo stroke model. They find that the fusion protein protects the brain against the effect of ischemia when applied before stroke onset. Here, a concise review of neuroglobin research and the application of CPP peptides in hypoxia and ischemia is provided.     

In vivo tracking of human adipose-derived stem cells labeled with ferumoxytol in rats with middle cerebral artery occlusion by magnetic resonance imaging

Ferumoxytol, an iron replacement product, is a new type of superparamagnetic iron oxide approved by the US Food and Drug Administration. Herein, we assessed the feasibility of tracking transplanted human adipose-derived stem cells labeled with ferumoxytol in middle cerebral artery occlusion-injured rats by 3.0 T MRI in vivo. 1 × 10⁴ human adipose-derived stem cells labeled with ferumoxytol-heparin-protamine were transplanted into the brains of rats with middle cerebral artery occlusion. Neurologic impairment was scored at 1, 7, 14, and 28 days after transplantation. T2-weighted imaging and enhanced susceptibility-weighted angiography were used to observe transplanted cells. Results of imaging tests were compared with results of Prussian blue staining. The modified neurologic impairment scores were significantly lower in rats transplanted with cells at all time points except 1 day post-transplantation compared with rats without transplantation. Regions with hypointense signals on T2-weighted and enhanced susceptibility-weighted angiography images corresponded with areas stained by Prussian blue, suggesting the presence of superparamagnetic iron oxide particles within the engrafted cells. Enhanced susceptibility-weighted angiography image exhibited better sensitivity and contrast in tracing ferumoxytol-heparin-protamine-labeled human adipose-derived stem cells compared with T2-weighted imaging in routine MRI.     

Post-ischemic administration of nimodipine following focal cerebral ischemic-reperfusion injury in rats alleviated excitotoxicity, neurobehavioural alterations and partially the bioenergetics

The present study focuses on the temporal calcium significance in middle cerebral artery occluded (2 h ischemia)-reperfused (70 h reperfusion) rats treated with nimodipine (NM) through concurrent measurements of excitotoxicity, bioenergetics and neurobehavioural paradigms. Further, the suitable therapeutic time window of calcium channel antagonism in stroke was also ascertained. NM (5 mg/kg, i.p.) was administered at pre (30 min before the induction of ischemia), during (1 h following occlusion of MCA) and post-ischemic (3 h after begin of reperfusion) states. The magnitude of neuroprotection in terms of excitotoxicity (glutamate, glutamine synthetase, Na⁺K⁺ATPase), bioenergetics (ATP, NAD⁺) and neurobehavioural paradigms (neurological score and open field exploratory behaviour) were measured and compared to ensure the therapeutic time-window of NM in stroke. Middle cerebral artery occlusion-reperfusion (MCAO/R) was found to elevate glutamate, glutamine synthetase levels and deplete Na⁺K⁺ATPase activity in the vehicle treated group (IR group). Significant decrease in bioenergetics such as ATP and NAD⁺ levels was also observed. Further, IR group demonstrated grievous oxidative stress (increase in lipid peroxidation, protein carbonyl content, nitrite/nitrate levels and decrease in superoxide dismutase and glutathione levels) along with anxiogenic behaviour, neurological deficits and neuronal damage and decreased nuclear to cytoplasm ratio in CA1 hippocampal region. Post-ischemic NM administration reversed the excitotoxicity, neurobehavioural and histopathological alterations significantly, but it restored bioenergetics level in MCAO/R rats only partially.These findings were further confirmed with the combination treatment (CT) of post-ischemic NM and pre-ischemic memantine (MN) administration, since MN showed protective effect in the pre-ischemic administration (Babu and Ramanathan, 2009). The failure of NM to forefend the neurodegeneration on pre- and during-ischemic administration suggests that the initial phase damages in ischemic-reperfusion (IR) might be mediated through other mechanism(s) such as glutamergic overstimulation or reverse operation of glutamate transporters. From the present study, it is concluded that calcium plays a crucial role in post-ischemic status and the suitable therapeutic time window of calcium antagonism is the post-ischemic state.     

N-(quinolin-2-ylmethyl)butane-1,4-diamine,a polyamine analogue,attenuated injury in in vitro and in vivo models of cerebral ischemia

It has been widely recognized that glutamate (Glu)-induced cytotoxicity, intracellular calcium overload and excessive free radical production are the key players in the development and progression of ischemic brain injury. Since MK-801, an antagonist of N-methyl-d-aspartate (NMDA) receptor, showed many adverse reactions that hampered its clinical applications, development of safe and effective agent for the treatment of cerebral ischemia is eagerly required. This study was to investigate the effects of N¹-(quinolin-2-ylmethyl)butane-1,4-diamine (QMA), a polyamine analogue, on the in vitro and in vivo models of cerebral ischemic damage. The results revealed that pretreatment with QMA could attenuate Glu, putrescine (Put) and oxygen-glucose deprivation (OGD)-induced cell death, lipid peroxidation as well as the elevation of reactive oxygen species (ROS) and intracellular [Ca²⁺]_i in pheochromocytoma (PC12) cells and in rat primary cortical neurons. The results also demonstrated that QMA could inhibit NMDA-mediated intracellular [Ca²⁺]_i accumulation in rat primary cortical neurons and reduce brain infarct volume in middle cerebral artery occlusion (MCAO) rats. The present report suggested that polyamines played a crucial role in the pathological processes of cerebral ischemic damage and that QMA or other novel polyamine analogues could be promising therapeutic candidates for stroke by virtue of their anti-hypoxia and antioxidation property.     

Total oligomeric flavonoids of Cyperus rotundus ameliorates neurological deficits, excitotoxicity and behavioral alterations induced by cerebral ischemic-reperfusion injury in rats

Interactions between neurons and astrocytes play a critical role in the central nervous system homeostasis. Cyperus rotundus (family: Cyperaceae), a traditional Indian medicinal herb, used as nervine tonic and nootropic in the Ayurvedic system of medicine. The present study was undertaken to investigate the neuroprotective effect of total oligomeric flavonoids (TOFs), prepared from C. rotundus, in rat model of cerebral ischemia and reperfusion. Male Sprague Dawley rats (290-340 g) were subjected to middle cerebral artery occlusion (MCAO) for 2 h and reperfusion for 70 h. Experimental animals were divided into four groups: Group I - sham operated (n = 7); Group II - vehicle treated ischemic-reperfusion (IR) (n = 9), and Group III and IV - TOFs treated (100 and 200 mg/kg body weight, p.o., respectively; n = 7 in each group). Vehicle or TOFs were pretreated for four days before the induction of ischemia and continued for next three days after the ischemia i.e. treatment was scheduled totally for a period of 7 days. MCAO surgery was performed on day 4, 1 h after TOFs administration. Neuroprotective effect of TOFs was substantiated in terms of neurological deficits, excitotoxicity (glutamate, glutamine synthetase and Na⁺K⁺ATPase levels), oxidative stress (malondialdehyde, super oxide dismutase, and glutathione) and neurobehavioral functions in the experimental animals. TOFs decreased glutamate, glutamine synthetase (GS) and increased Na⁺K⁺ATPase activity in a dose dependent manner when compared to the IR rats. Treatment with TOFs significantly reduced the neurological deficits and reversed the anxiogenic behavior in rats. Further, it also significantly decreased MDA and increased superoxide dismutase (SOD) and glutathione content in brains of experimental rats. Histopathological examination using cresyl violet staining revealed the attenuation of neuronal loss by TOFs in stroke rats. The present study demonstrates the unswerving involvement of TOFs on ischemia-reperfusion triggered biochemical alterations in MCAO/R rats. Hence, TOFs might be an attractive candidate for further studies in the development of new drugs for cerebral stroke treatment.     

Reduction of Ischemia Reperfusion-Related Brain Hemorrhage by Stachybotrys Microspora Triprenyl Phenol-7 in Mice With Antioxidant Effects

Background

Stachybotrys microspora triprenyl phenol-7 (SMTP-7) has both thrombolytic and anti-inflammatory effects, but its neuroprotective effects on cerebral ischemia are still unclear. The present study assessed the antioxidative and neurovascular unit (NVU) protective effects of SMTP-7 using transient middle cerebral artery occlusion (tMCAO) mice.