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C57BL/6 strain is most susceptible to cerebral ischemia following bilateral common carotid occlusion among seven mouse strains: Selective neuronal death in the murine transient forebrain ischemia
Abstract
Rats and gerbils have been used widely to investigate the molecular mechanism of selective neuronal death following transient global ischemia. Recently, the availability of transgenic mice has enabled us to examine the involvement of specific gene products in various pathophysiological conditions. However, there has been only limited information about the experimental model of cerebral ischemia in mice, particularly in regard to selective neuronal death. We examined whether bilateral carotid occlusion produced global forebrain ischemia in seven common mouse strains including C57BL/6, ICR, BALB/c, C3H, CBA, ddY and DBA/2, based on neurological signs, histological findings and cortical microcirculatory as well as India ink perfusion patterns. The C57BL/6 strain was found to be the most susceptible among seven strains. All C57BL/6 mice died within 6 h after permanent bilateral carotid occlusion. After transient bilateral carotid occlusion for 20 min, more than 90% of C57BL/6 mice showed typical neurological signs such as torsion of the neck and rolling fits, and developed selective neuronal death in the hippocampus and caudoputamen. Hypothermia prevented the neuronal death. Visualization of brain vasculature by India ink perfusion indicated that the susceptibility of the mice after bilateral carotid occlusion depended mainly on the degree of anastomosis between carotid and basilar arteries. Our results showed the feasibility of investigating selective neuronal death in transgenic mice with simple temporary occlusion of both common carotid arteries, when those from the C57BL/6 strain or inbred transgenic mice from other strains with the C57BL/6 strain in a back-cross manner are used.
... It is associated with a huge range of clinical ocular disorders and diseases such as diabetic retinopathy, glaucoma, optic neuropathies and stroke (Dattilo, Newman & Biousse, 2018;Fouda et al., 2020;Minhas, Morishita & Anand, 2012;Osborne et al., 2004). As oxygen (directly referred to as ''blood'') in the retina is supplied through the ophthalmic artery originated from the internal carotid artery, one of two branches of the common carotid artery (CCA), animal models by common carotid artery occlusion or stenosis (CCAO or CCAS) have been used to understand the pathogenesis of retinal ischemia (Crespo-Garcia et al., 2018;Davidson et al., 2000;Lavinsky et al., 2006;Lee et al., 2020b;Qin et al., 2019;Sun et al., 2019;Venkat, Chopp & Chen, 2015;Wellons Iii et al., 2000;Yamamoto et al., 2006;Yang et al., 1997). ...
... Therefore, CCAO or CCAS has been attempted to develop animal models for such retinal ischemic diseases. However, several previous studies demonstrated that experimental models (mice and rats) could die during and after CCAO or CCAS, in that severe brain damages as well as cardiac arrests were induced by the operation (Crespo-Garcia et al., 2018;Venkat, Chopp & Chen, 2015;Wellons Iii et al., 2000;Yang et al., 1997). This evokes several matters such as unexpected loss and individual variations of experimental models. ...
... In this regard, we previously established a stable mouse model of retinal ischemia induced by unilateral CCAO (UCCAO) with 100% survival rate during and after the operation through minimization of brain damages and showed time courses of common ischemic findings in the unilateral retina (Lee et al., 2020a), which were generally seen in other CCAO and CCAS models (Crespo-Garcia et al., 2018;Davidson et al., 2000;Lavinsky et al., 2006;Lee et al., 2020b;Ogishima et al., 2011;Qin et al., 2019;Sun et al., 2019;Venkat, Chopp & Chen, 2015;Wellons Iii et al., 2000;Yamamoto et al., 2006;Yang et al., 1997). However, time courses of retinal functional changes and acute retinal injuries in this model have not been clearly studied. ...
Background:
Retinal ischemic stresses are associated with the pathogenesis of various retinal vascular diseases. To investigate pathological mechanisms of retinal ischemia, reproducible, robust and clinically significant experimental rodent models are highly needed. Previously, we established a stable murine model of chronic hypoperfusion retinal injuries by permanent unilateral common carotid artery occlusion (UCCAO) and demonstrated chronic pathological processes in the ischemic retina after the occlusion; however, retinal functional deficits and other acute retinal ischemic injuries by UCCAO still remain obscure. In this study, we attempted to examine retinal functional changes as well as acute retinal ischemic alterations such as retinal thinning, gliosis and cell death after UCCAO.
Methods:
Adult mice (male C57BL/6, 6-8 weeks old) were subjected to UCCAO in the right side, and retinal function was primarily measured using electroretinography for 14 days after the surgery. Furthermore, retinal thinning, gliosis and cell death were investigated using optical coherence tomography, immunohistochemistry and TUNEL assay, respectively.
Results:
Functional deficits in the unilateral right retina started to be seen 7 days after the occlusion. Specifically, the amplitude of b-wave dramatically decreased while that of a-wave was slightly affected. 14 days after the occlusion, the amplitudes of both waves and oscillatory potentials were significantly detected decreased in the unilateral right retina. Even though a change in retinal thickness was not dramatically observed among all the eyes, retinal gliosis and cell death in the unilateral right retina were substantially observed after UCCAO.
Conclusions:
Along with previous retinal ischemic results in this model, UCCAO can stimulate retinal ischemia leading to functional, morphological and molecular changes in the retina. This model can be useful for the investigation of pathological mechanisms for human ischemic retinopathies and furthermore can be utilized to test new drugs for various ischemic ocular diseases.
... It has been reported in the past that the C57BL/6 strain is highly susceptible to cerebral ischemia when compared to other strains such as the ICR, BALB/c, and C3H strains [20]. Previous studies also showed that severe ischemia occurs in the C57BL/6 strain when bilateral CCA occlusion was performed by temporarily occluding the carotid artery using microaneurysm clips for 20 minutes [20]. The poorly developed or even absent posterior communicating artery in close to 90% of the C57BL6/J mice [8,21] could have accounted for this high ...
... In comparison to a previously reported study [7] where the survival rate of the VCI-induced mice was around 80% at post 28 days, the survival rate for the current study was around 27%. It has been reported in the past that the C57BL/6 strain is highly susceptible to cerebral ischemia when compared to other strains such as the ICR, BALB/c, and C3H strains [20]. Previous studies also showed that severe ischemia occurs in the C57BL/6 strain when bilateral CCA occlusion was performed by temporarily occluding the carotid artery using microaneurysm clips for 20 minutes [20]. ...
... It has been reported in the past that the C57BL/6 strain is highly susceptible to cerebral ischemia when compared to other strains such as the ICR, BALB/c, and C3H strains [20]. Previous studies also showed that severe ischemia occurs in the C57BL/6 strain when bilateral CCA occlusion was performed by temporarily occluding the carotid artery using microaneurysm clips for 20 minutes [20]. The poorly developed or even absent posterior communicating artery in close to 90% of the C57BL6/J mice [8,21] could have accounted for this high susceptibility. ...
Recently, an asymmetric vascular compromise approach that replicates many aspects of human vascular cognitive impairment (VCI) has been reported. The present study aimed to first investigate on the reproducibility in the disease progression of this newly reported VCI model using wild-type C57BL6/J mice. The second aim was to assess how this approach will affect the disease progression of transgenic Alzheimer’s disease (AD) 5XFAD mice subjected to VCI. C57BL6/J and 5XFAD mice were subjected to VCI by placing an ameroid constrictor on the right CCA and a microcoil on the left CCA. Infarcts and hippocampal neuronal loss did not appear predominantly in the right (ameroid side) as expected but randomly in both hemispheres. The mortality rate of C57BL6/J mice was unexpectedly high. Inducing VCI reduced amyloid burden in the hippocampi of 5XFAD mice. Since VCI is known to be complex and complicated, the heterogeneous disease progression observed from this current study shares close resemblance to the clinical manifestation of VCI. This heterogeneity, however, makes it challenging to test novel treatment options using this model. Further study is warranted to tackle the heterogeneous nature of VCI.
... BCCAo models have been developed and widely used for global ischemia studies in gerbils, 22,23 rats 13,24 and mice. 7,[25][26][27] The lack of communication between the anterior and posterior cerebral circulations observed in gerbils has allowed the use of these animals to induce tGCI. They are vulnerable to neurological deficits and brain damage with a simply and short occlusion of the carotids at the neck 22 even as short as 5 minutes. ...
... As stated above and in several studies, mouse strains exhibit diverse levels of vulnerability to cerebral ischemia that has to be consider by the researchers. 25,33,34 The first study of strain divergences reported a higher sensitivity of BALB/c mice to ischemia over BDF (a strain generated from female C57 and male DBA 2 mice) and CFW (an outbred strain, originated from inbreeding Swiss mice) strains that was not attributable to main arterial blood pressure (MABP) or blood gases, due to the anatomy of the circle of Willis. The absence of patent posterior communicating arteries in BALB/c was evident using a carbon black injection and showed this strain as the most suitable to induce a global brain ischemia. ...
... 33 Another study by Wellons and coworkers, revealed not significant CA1 damage in SV-129 mice compared with C57BL/6 regardless the type of ischemic injury (BCCAo alone or with hypotension), exposing also resistance of SV-129 mice to global ischemia; however, they conclude that there are insufficient data to associate vascular anatomy with these susceptibility differences. 38 Nevertheless, the most vulnerable strain to ischemia followed BCCAo is the C57BL/6, because it shows reliable neurological signs of ischemia and highly reproducible histological damage in the hippocampus and caudo-putamen, as observed in the work of Yang and coworkers (1997) in six different mouse strains,C57BL/6, ICR, BALB/c, DBA, CBA/J, C3H, and ddY, in which DBA and ddY strains showed a better resistant to occlusion of 24 hours; 25 while in other studies, the MF1 39 and SV-129 36 mouse strains, a lower ischemia vulnerability after carotid occlusion was observed compared to C57BL/6, because of differences in vascular and arterial blood pressure. In addition of high mortality rate and histological damage in C57BL/6, hippocampal neuronal death was not homogeneous between individuals or hemispheres of the same animal since some CA1, CA2 and CA3 regions of hippocampus were destroyed while others showed damage only in CA2 or medial CA1 as well as few animals showed bilateral symmetric injury. ...
Brain ischemia is one of the principal causes of death and disability worldwide in which prevention or an effective treatment does not exist. In order to develop successful treatments, an adequate and useful ischemia model is essential. Transient global cerebral ischemia is one of the most interesting pathological conditions in stroke studies because of the observed degeneration of forebrain and delayed neuronal cell death in selective vulnerable regions such as hippocampus. Transient occlusion of both common carotid arteries is the most convenient model to induce tGCI. Although there are effective rat and gerbil models using this method, the induction of a reproducible and reliable injury after global ischemia in mouse has presented higher variations, mainly because of its size and the necessary monitoring skills in order to accomplish homogeneous and reproducible results. Further, great variability among cerebral vasculature and susceptibility of the different strains and sub-strains is observed. In recent years, some modifications have been made to the model in order to normalize the heterogenic effects. Analysis of posterior communicating artery patency has been proposed as an exclusion parameter due to the direct relationship reported with the reduction of cerebral blood flow. Another method used to significantly reduce blood flow is the induction of hypotension with isoflurane. Each protocol produces distinct injury outcomes. Further improvements are needed to attain a general, simpler, reproducible and globally accepted model that allows comparisons between research groups, progress in understanding ischemia and the consequent development of therapeutic alternatives for ischemic injury.
... The diameter and number of PComs (i.e., whether they are present bilaterally, unilaterally, or absent) also appear to vary widely in different mouse strains, suggesting involvement of genetic factors [39][40][41][42][43][44][45][46][47][48]. However, there is considerable disagreement among studies even for the same strain. ...
... The PCom diameter and number did not differ with sex in B6 mice, although there was a trend for larger diameter and number in females [69]. The mouse PCom, as defined herein, is in the anatomic location of the P1 segment of the PCA in humans (visa versa for the human PCom) (see Fig. 1)-definitions based on diameter and function that are also used in most previous mouse studies, e.g., [41,42]. Angiography was performed after maximal dilation, fixation, and filling with latex. ...
... Earlier reports employing India ink angiography suggested that PCom variation exists among different strains of mice; however, significant disagreement exists among the reports (discussed below). As well, comparisons with our findings are complicated since previous studies did not use perfusion fixation at maximal dilation, often lacked quantification, relied on smaller n sizes, and in several studies [38][39][40][41] did not differentiate among unilateral, bilateral, or absent PComs. Thus, PComs were reportedly Bintact^(bilateral) in 10 each of B6/J, BALB/cCF, and 129/J mice [42], lacked Bgross differences^in B6 and 129/J [43], and were Bbilateral and hypoplastic^(small diameter) in B6/Tac versus Bwell formedî n 129Sv/Tac [44]. ...
Variation in blood flow mediated by the posterior communicating collateral arteries (PComs) contributes to variation in the severity of tissue injury in obstructive disease. Evidence in animals and humans indicates that differences in the extent of PComs, i.e., their anatomic lumen diameter and whether they are present bilaterally, unilaterally, or absent, are a major factor. These differences arise during development since they are present at birth. However, the causal mechanisms are unknown. We used angiography after maximal dilation to examine involvement of genetic, environmental, and stochastic factors. The extent of PComs varied widely among seven genetically diverse strains of mice. Like pial collaterals in the microcirculation, aging and hypertension reduced PCom diameter, while in contrast, obesity, hyperlipidemia, metabolic syndrome, and diabetes mellitus had no effect. Naturally occurring intrauterine growth restriction had no effect on extent of PCom or pial collaterals in the adult. The number and diameter of PComs evidenced much larger apparent stochastic-dependent variation than pial collaterals. In addition, both PComs underwent flow-mediated outward remodeling after unilateral permanent MCA occlusion that varied with genetic background and was greater on the ipsilesional side. These findings indicate that variation in the number and diameter of PCom collateral arteries arises from stochastic factors and naturally occurring genetic variants that differ from those that cause variation in pial collateral arterioles. Environmental factors also contribute: aging and hypertension reduce PCom diameter. Our results suggest possible sources of variation of PComs in humans and provide information relevant when studying mouse models of occlusive cerebrovascular disease.
... We previously observed that RECK immunoreactivity was low in adult mouse cerebrum but was increased in certain regions after transient cerebral ischemia (Wang et al., 2010). In mice subjected to common carotid artery occlusion (CCAO) (Yang et al., 1997) for 1 h followed by reperfusion, round RECK-positive cells (with a character of neural precursor cells) emerged in the cornus ammonis 2 (CA2) region of the hippocampus at day 2; at day 7, the RECK-positive cells increased in number, extended processes, and were distributed widely in the hippocampus ( Figure 3a, b) (Wang et al., 2010). Those findings raised the question as to whether the round RECK-positive cells in CA2 proliferate and migrate into wider areas (Model 1, Figure 3c) or some CA2 cells express RECK quickly after ischemia, while the cells in the other areas begin to express RECK at later stages (Model 2, Figure 3d). ...
... CCAO was performed as described previously (Wang et al., 2010;Yang et al., 1997). In brief, male mice (8-weeks old) anesthetized with Avertin and kept warm at 378C (with a homeothermic blanket) were subjected to bilateral common carotid artery occlusion (CCAO) for 1 h followed by reperfusion as described (Yang et al. 1997). ...
... CCAO was performed as described previously (Wang et al., 2010;Yang et al., 1997). In brief, male mice (8-weeks old) anesthetized with Avertin and kept warm at 378C (with a homeothermic blanket) were subjected to bilateral common carotid artery occlusion (CCAO) for 1 h followed by reperfusion as described (Yang et al. 1997). For histological examinations, brain tissues were fixed in 1% p-formaldehyde in 0.1M phosphate buffer (PB, pH 5 7.2). ...
Reck encodes a membrane-anchored glycoprotein implicated in the regulation of extracellular metalloproteinases, Notch-signaling, and Wnt7-signaling and shown to play critical roles in embryogenesis and tumor suppression. Precise mechanisms of its actions in vivo, however, remain largely unknown. By homologous recombination, we generated a new Reck allele, ReckCreERT2 (MGI symbol: Reck<tm3.1(cre/ERT2)Noda>). This allele is defective in terms of Reck function but expected to induce loxP-mediated recombination in the cells committed to express Reck. Similarity in the expression patterns of the ReckCreERT2 transgene and the endogenous Reck gene was confirmed in five tissues. In the adult hippocampus, induction of Reck expression after transient cerebral ischemia could be demonstrated using this allele. These results indicate the utility of this Cre-driver allele in further studies. This article is protected by copyright. All rights reserved.
... We have used C57BL/6 mice, as our previous study [16] along with others [26,27] showed transient BCCAO in these mice developed consistent and selective neuronal death in the CA1, CA2 and CA3 areas of the hippocampus and motor, somatosensory and insular cortex with a higher survival rate [16,26,27]. The reduction in the neuronal cell number in the stroke group was due to the high sensitivity of neurons to ischaemic effects and the blood supply reduction, causing neuronal death in these brain areas [28]. ...
... We have used C57BL/6 mice, as our previous study [16] along with others [26,27] showed transient BCCAO in these mice developed consistent and selective neuronal death in the CA1, CA2 and CA3 areas of the hippocampus and motor, somatosensory and insular cortex with a higher survival rate [16,26,27]. The reduction in the neuronal cell number in the stroke group was due to the high sensitivity of neurons to ischaemic effects and the blood supply reduction, causing neuronal death in these brain areas [28]. ...
The blood-brain barrier (BBB) acts as a specialized structure separating the brain from peripheral blood circulation and plays an important role in brain function. Following an ischaemic stroke or cerebral ischaemia, the BBB is damaged leading to degraded proteins being released into blood circulation. However, little is known about cerebral ischaemia and reperfusion (I/R) induced BBB damage and changes in circulatory biomarkers. This study aims to use both immunohistochemistry and western blotting (WB) to examine neuronal death, glial cell alterations and changes in BBB tight junction (TJ) proteins such as zonula occludens-1 (ZO-1), Occludin and Claudin-5 in the hippocampus in a murine model of cerebral I/R. The changes in these proteins in the blood serum of this model were assessed by enzyme-linked immunosorbent assay. The results showed neuronal death and a significant increase in glial fibrillary acidic protein (GFAP), a protein primarily expressed in astrocytes and a significant decrease in TJ proteins, ZO-1, Occludin and Claudin-5 in the hippocampus of occluded mice as compared to sham-operated mice. These changes are associated with an increased level of these proteins in blood serum in ischaemic mice, suggesting that these proteins can be used as potential biomarkers for determining ischaemic stroke.
... Using bilateral occlusions of CCAs, rats have been widely used for understanding the pathological mechanism for retinal degeneration in OIS [8][9][10]. Relatively, mice have not been commonly utilized, as there exist the limitations for the model development using bilateral occlusions of CCAs [11,12]. Bilateral stenosis of CCAs was attempted in mice using tricky microcoils under limited conditions such as probably excluding dead mice during/after the surgery [13]. ...
... Then, how about the other strains of mice? It would be interesting to perform a comparison study on ocular ischemic stresses by UCCAO in different strains of mice, as similar as a comparison study conducted in 1997 on cerebral ischemic stresses by BCCAO in several mouse strains [12]. ...
Retinal degeneration is a progressive retinal damage in ocular vascular diseases. There are several reasons for this, such as occlusion of arteries or veins, diabetic retinopathy, or hereditary retinal diseases. To study pathological mechanisms of retinal degeneration, it is required to develop experimentally reproducible and clinically relevant models. In our previous studies, we developed a murine model of retinal hypoperfusion by unilateral common carotid artery occlusion (UCCAO) which mimics the pathophysiology of ocular ischemic syndrome (OIS) in humans, and described broad pathological mechanisms in the retina after UCCAO. However, there still remain missing pieces of the ocular pathologic process by UCCAO. In this study, we examined those unfound mechanisms. UCCAO was performed on adult mice. Ocular dysfunctions, histological deficits, and inflammation were examined after UCCAO, compared with sham-operated mice. Evaluation values were analyzed by electrophysiological, histological, and molecular biological methods. Eyelid drooping was permanently seen after UCCAO. Induction time point of acute reversible cataract under anesthesia was shortened. Retinal/visual dysfunctions were detected 2-4 weeks after UCCAO. Specifically, scotopic b-wave was more affected than a-wave, with the dysfunction of photopic b-wave. Impaired oscillatory potentials and visual evoked potential were constantly observed. Pathological Müller gliosis/inflammation was featured with NeuN-positive cell loss in the ganglion cell layer. Axial length, intraocular pressure, pupillary light reflex, and retinal pigment epithelium/choroidal thickness were not changed by UCCAO. A murine model of retinal ischemia by UCCAO can be useful for studying a series of degenerative process in the ischemic retina.
... Often, the variance in lesion size, which is one of the main outcome parameters, is as high as 40% of the mean, rendering the generation of robust and reproducible data with prevalently used small sample sizes difficult (Dirnagl, 2006). The extent of this variability greatly depends on the mouse strain used in the experiment (Yang et al., 1997;Kitagawa et al., 1998). One of the most commonly used mouse strains, but at the same time one of the strains with the largest variability in lesion size, is C57BL/6, due to its widespread use for generating genetically modified mice (Kitagawa et al., 1998;Maeda et al., 1998;McColl et al., 2004). ...
... One of its inherent disadvantages is the high intersubject variability of common outcome parameters. Several variables have been shown to impact lesion volume, one of the most important outcome parameters (Morris et al., 2016); including different parameters of the model itself, such as the occlusion and recovery time (Harada et al., 2005;Liu et al., 2009), the filament and anesthetic used (Michenfelder et al., 1976;Tsuchiya et al., 2003), differences in the surgical technique (Chen et al., 2008) as well as parameters of the animals, such as the species (Carmichael, 2005), strain (Yang et al., 1997;Kitagawa et al., 1998), substrain (Zhao et al., 2019), sex (Alkayed et al., 1998;Zhao et al., 2019), age (Sutherland et al., 1996), and interindividual variation in cerebrovascular anatomy (Barone et al., 1993;Kitagawa et al., 1998;McColl et al., 2004). Species and strain differences in the cerebrovascular architecture and in particular differences in the patency of macroanastomoses formed in the circle of Willis have already been studied previously (Barone et al., 1993;Kitagawa et al., 1998;McColl et al., 2004). ...
Numerous studies on experimental ischemic stroke use the filament middle cerebral artery occlusion (fMCAo) model in C57BL/6 mice, but lesion sizes in this strain are highly variable. A known contributor is variation in the posterior communicating artery (PcomA) patency. We therefore aimed to provide a semiquantitative non-invasive in vivo method to routinely assess PcomA patency. We included 43 male C57BL/6 mice from four independent studies using a transient 45 min fMCAo model. Edema-corrected lesion sizes were measured by magnetic resonance (MR) imaging 24 h after reperfusion. Time-of-flight MR angiography was performed 7 days before and 24 h after fMCAo. Scores of PcomA size measured 24 h after, but not scores measured 7 days before fMCAo were negatively correlated with lesion size. Variability in PcomA patency explained 30% of the variance in our cohort (p < 0.0001, coefficient of determination r² = 0.3). In a simulation using parameters typical for experimental stroke research, the power to detect a true effect of d = 1 between two groups increased by 15% when an according covariate was included in the statistical model. We have demonstrated that in vivo measurement of PcomA size is feasible and can lead to increased accuracy in assessing the effect of treatments.
... There is a possibility that the optic tract in FVB/C57Bl/6J F1 mice is more vulnerable to hypoperfusion than C57Bl/6J mice due to genetic differences. It is also reported that the cerebral vasculature of different strains is differently organised and may therefore present with different spatial distribution and severity of hypoperfusion 56 . The heightened vulnerability of the optic tract may also account for the impaired behaviour in the radial arm maze in the hypoperfused mice. ...
... In the current study, both micro-and astrogliosis were increased, however only in the optic tract of hypoperfused mice. The regional discrepancy between this study and previous may be attributed to the background strain, which as previously mentioned, may respond to hypoperfusion differently due to do variances in cerebrovascular arrangement 56 . Overexpression of Nrf2 reduced the extent of astrogliosis, but did not have an effect on microgliosis after hypoperfusion. ...
Mouse models have shown that cerebral hypoperfusion causes white matter disruption and memory impairment relevant to the study of vascular cognitive impairment and dementia. The associated mechanisms include inflammation and oxidative stress are proposed to drive disruption of myelinated axons within hypoperfused white matter. The aim of this study was to determine if increased endogenous anti-oxidant and anti-inflammatory signalling in astrocytes was protective in a model of mild cerebral hypoperfusion. Transgenically altered mice overexpressing the transcription factor Nrf2 (GFAP-Nrf2) and wild type littermates were subjected to bilateral carotid artery stenosis or sham surgery. Behavioural alterations were assessed using the radial arm maze and tissue was collected for pathology and transcriptome analysis six weeks post-surgery. GFAP-Nrf2 mice showed less pronounced behavioural impairments compared to wild types following hypoperfusion, paralleled by reduced optic tract white matter disruption and astrogliosis. There was no effect of hypoperfusion on anti-oxidant gene alterations albeit the levels were increased in GFAP-Nrf2 mice. Instead, pro-inflammatory gene expression was determined to be significantly upregulated in the optic tract of hypoperfused wild type mice but differentially affected in GFAP-Nrf2 mice. In particular, complement components (C4 and C1q) were increased in wild type hypoperfused mice but expressed at levels similar to controls in hypoperfused GFAP-Nrf2 mice. This study provides evidence that overexpression of Nrf2 in astrocytes exerts beneficial effects through repression of inflammation and supports the potential use of Nrf2-activators in the amelioration of cerebrovascular-related inflammation and white matter degeneration.
... Transient Global Cerebral Ischemia Female C57BL/6 mice (18-22 g) were subjected to BCCAO surgery to induce transient global cerebral ischemia as previously reported [18][19][20][21]. The C57BL/6 strain is reported to be more susceptible to BCCAO-induced global ischemia than other strains due to poorly developed posterior communicating arteries [22,23]. Anesthesia was induced with inhalation of 2% isoflurane in a mixture of air and oxygen (70:30) and maintained with 1.5% isoflurane during the surgery period. ...
... The common carotid arteries were exposed via a neck incision and encircled with loose ligatures for later clamping. The BCCAO was induced by applying micro-serrefine clamps (Fine Science Tools, CA) to both common carotid arteries for 20 min, determined by our preliminary studies and as previously reported [20,21,23]. After releasing the clamps, the skin incision was closed using Reflex Wound Clips (Fine Science Tools, CA). ...
Cerebral ischemia leads to multifaceted injury to the brain. A polytherapeutic drug that can be administered immediately after reperfusion may increase protection to the brain by simultaneously targeting multiple deleterious cascades. This study evaluated efficacy of the combination of three clinically approved drugs: lamotrigine, minocycline, and lovastatin, using two mouse models: global and focal cerebral ischemia induced by transient occlusion of the common carotid arteries or the middle cerebral artery, respectively. In vitro, the combination drug, but not single drug, protected neurons against oxygen-glucose deprivation (OGD)-induced cell death. The combination drug simultaneously targeted cell apoptosis and DNA damage induced by ischemia. Besides acting on neurons, the combination drug suppressed inflammatory processes in microglia and brain endothelial cells induced by ischemia. In a transient global ischemia model, the combination drug, but not single drug, suppressed microglial activation and inflammatory cytokine production, and reduced neuronal damage. In a transient focal ischemia model, the combination drug, but not single drug, attenuated brain infarction, suppressed infiltration of peripheral neutrophils, and reduced neurological deficits following ischemic stroke. In summary, the combination drug confers a broad-spectrum protection against ischemia/reperfusion (I/R) injury and could be a promising approach for early neuroprotection after out-of-hospital cardiac arrest or ischemic stroke.
... The forebrain IR model resembles the temporary bilateral common carotid artery ligation (BCCAL) model [17]. Because blood flow is decreased in the frontal, parietal, and temporal lobes of the brain during the ischemic period, although the cerebellum is still perfused without the basilar artery closing, demonstrating a patent and well-established posterior collateral supply, it serves as a model for forebrain IR injury [3]. ...
Citation: Marghani, B.H.; Rezk, S.; Ateya, A.I.; Alotaibi, B.S.; Othman, B.H.; Sayed, S.M.; Alshehri, M.A.; Shukry, M.; Mansour, M.M. The Effect of Cerebrolysin in an Animal Abstract: Forebrain ischemia-reperfusion (IR) injury causes neurological impairments due to decreased cerebral autoregulation, hypoperfusion, and edema in the hours to days following the restoration of spontaneous circulation. This study aimed to examine the protective and/or therapeutic effects of cerebrolysin (CBL) in managing forebrain IR injury and any probable underlying mechanisms. To study the contribution of reperfusion to forebrain injury, we developed a transient dual carotid artery ligation (tDCAL/IR) mouse model. Five equal groups of six BLC57 mice were created: Group 1: control group (no surgery was performed); Group 2: sham surgery (surgery was performed without IR); Group 3: tDCAL/IR (surgery with IR via permanently ligating the left CA and temporarily closing the right CA for 30 min, followed by reperfusion for 72 h); Group 4: CBL + tDCAL/IR (CBL was given intravenously at a 60 mg/kg BW dose 30 min before IR); and Group 5: tDCAL/IR + CBL (CBL was administered i.v. at 60 mg/kg BW three hours after IR). At 72 h following IR, the mice were euthanized. CBL administration 3 h after IR improved neurological functional recovery, enhanced anti-inflammatory and antioxidant activities, alleviated apoptotic neu-ronal death, and inhibited reactive microglial and astrocyte activation, resulting in neuroprotection after IR injury in the tDCAL/IR + CBL mice group as compared to the other groups. Furthermore, CBL reduced the TLRs/NF-kB/cytokines while activating the Keap1/Nrf2/antioxidant signaling pathway. These results indicate that CBL may improve neurologic function in mice following IR.
... Both mice strains were chosen because of patent posterior communicating arteries in the circle of Willis. 17 The presence of the posterior communicating artery in the mice used in this particular study was confirmed by injecting dye into the aorta after sacrifice and anatomical preparation of the circle of Willis (figure 1A). ...
Objective
One of the most important risk factors for developing a glaucomatous optic neuropathy is elevated intraocular pressure. Moreover, mechanisms such as altered perfusion have been postulated to injure the optical path. In a mouse model, we compare first negative effects of cerebral perfusion/reperfusion on the optic nerve structure versus alterations by elevated intraocular pressure. Second, we compare the alterations by isolated hypoperfusion-reperfusion and isolated intraocular pressure to the combination of both.
Methods and analysis
Mice were divided in four groups: (1) controls; (2) perfusion altered mice that underwent transient bi-common carotid artery occlusion (BCCAO) for 40 min; (3) glaucoma group (DBA/2J mice); (4) combined glaucoma and altered perfusion (DBA/2J mice with transient BCCAO). Optic nerve sections were stereologically examined 10–12 weeks after intervention.
Results
All experimental groups showed a decreased total axon number per optic nerve compared with controls. In DBA/2J and combined DBA/2J & BCCAO mice the significant decrease was roughly 50%, while BCCAO leaded to a 23% reduction of axon number, however reaching significance only in the direct t-test. The difference in axon number between BCCAO and both DBA/2J mice was almost 30%, lacking statistical significance due to a remarkably high variation in both DBA/2J groups.
Conclusion
Elevated intraocular pressure in the DBA/2J mouse model of glaucoma leads to a much more pronounced optic nerve atrophy compared with transient forebrain hypoperfusion and reperfusion by BCCAO. A supposed worsening effect of an altered perfusion added to the pressure-related damage could not be detected.
... Rats are a suitable rodent species as they have the complete circle of Willis (CoW) [35][36][37], which means that rats may not suddenly die after BCCAO. In contrast, the inadequate development of posterior communicating arteries in CoW causes dramatic cerebral ischemia in mice [38][39][40], finally leading to sudden death during/after BCCAO [29,[41][42][43]. ...
Ocular ischemic syndrome (OIS) is one of the severe ocular disorders occurring from stenosis or occlusion of the carotid arteries. As the ophthalmic artery is derived from the branch of the carotid artery, stenosis or occlusion of the carotid arteries could induce chronic ocular hypoperfusion, finally leading to the development of OIS. To date, the pathophysiology of OIS is still not clearly unraveled. To better explore the pathophysiology of OIS, several experimental models have been developed in rats and mice. Surgical occlusion or stenosis of common carotid arteries or internal carotid arteries was conducted bilaterally or unilaterally for model development. In this regard, final ischemic outcomes in the eye varied depending on the surgical procedure, even though similar findings on ocular hypoperfusion could be observed. In the current review, we provide an overview of the pathophysiology of OIS from various experimental models, as well as several clinical cases. Moreover, we cover the status of current therapies for OIS along with promising preclinical treatments with recent advances. Our review will enable more comprehensive therapeutic approaches to prevent the development and/or progression of OIS.
... CCH by permanent bilateral CCA ligation offers several advantages compared to other approaches. It can induce CCH in a more clinically relevant manner, without ischemic lesions in the brain [24,25]. The present study induced CCH by CCA ligation in the rat brain using real time CBF monitoring, and there was no ischemic lesion in the brain. ...
Background
Chronic cerebral hypoperfusion (CCH) is known to induce Alzheimer’s disease (AD) pathology, but its mechanism remains unclear. The purpose of this study was to identify the cerebral regions that are affected by CCH, and to evaluate the development of AD pathology in a rat model of CCH.
Methods
A rat model of CCH was established by bilaterally ligating the common carotid arteries in adult male rats (CCH group). The identical operations were performed on sham rats without arteries ligation (control group). Regional cerebral glucose metabolism was evaluated at 1 and 3 months after bilateral CCA ligation using positron emission tomography with F-18 fluorodeoxyglucose. The expression levels of amyloid β40 (Aβ40), amyloid β42 (Aβ42), and hyperphosphorylated tau were evaluated using western blots at 3 months after the ligation. Cognitive function was evaluated using the Y-maze test at 3 months after the ligation.
Results
At 1 month after the ligation, cerebral glucose metabolism in the entorhinal, frontal association, motor, and somatosensory cortices were significantly decreased in the CCH group compared with those in the control group. At 3 months after the ligation, cerebral glucose metabolism was normalized in all regions except for the anterodorsal hippocampus, which was significantly decreased compared with that of the control group. The expression of Aβ42 and the Aβ42/40 ratio were significantly higher in the CCH group than those in the control group. The phosphorylated-tau levels of the hippocampus in the CCH group were significantly lower than those in the control group. Cognitive function was more impaired in the CCH group than that in the control group.
Conclusion
Our findings suggest that CCH causes selective neurodegeneration of the anterodorsal hippocampus, which may be a trigger point for the development of AD pathology.
... Thus, Nestin-GFP mice (CB-17 background) from more than six generations acquired the im-portant traits of CB-17 wild-type mice. Previous studies have shown that C57BL/6 strain mice have a wide range of ischemic areas other than the cerebral cortex, including the striatum [3,17,[24][25][26]. The Nestin-GFP mice (C57BL/6 background) in the present study displayed a similar trend for their ischemic areas; therefore, a wide range of brain damage (e.g., ischemia and edema), which frequently occurs in strains with a C57BL/6 background, may be associated with the high mortality observed in these mice. ...
An accumulation of evidence shows that endogenous neural stem/progenitor cells (NSPCs) are activated following brain injury such as that suffered during ischemic stroke. To understand the expression patterns of these cells, researchers have developed mice that express an NSPC marker, Nestin, which is detectable by specific reporters such as green fluorescent protein (GFP), i.e., Nestin-GFP mice. However, the genetic background of most transgenic mice, including Nestin-GFP mice, comes from the C57BL/6 strain. Because mice from this background strain have many cerebral arterial branches and collateral vessels, they are accompanied by several major problems including variable ischemic areas and high mortality when subjected to ischemic stroke by occluding the middle cerebral artery (MCA). In contrast, CB-17 wild-type mice are free from these problems. Therefore, with the aim of overcoming the aforementioned defects, we first crossed Nestin-GFP mice (C57BL/6 background) with CB-17 wild-type mice and then developed Nestin-GFP mice (CB-17 background) by further backcrossing the generated hybrid mice with CB-17 wild-type mice. Subsequently, we investigated the phenotypes of the established Nestin-GFP mice (CB-17 background) following MCA occlusion; these mice had fewer blood vessels around the MCA compared with the number of blood vessels in Nestin-GFP mice (C57BL/6 background). In addition, TTC staining showed that infarcted volume was variable in Nestin-GFP mice (C57BL/6 background) but highly reproducible in Nestin-GFP mice (CB-17 background). In a further investigation of mice survival rates up to 28 days after MCA occlusion, all Nestin-GFP mice (CB-17 background) survived the period, whereas Nestin-GFP mice (C57BL/6 background) frequently died within 1 week and exhibited a higher mortality rate. Immunohistochemistry analysis of Nestin-GFP mice (CB-17 background) showed that GFP+ cells were mainly obverted in not only conventional neurogenic areas, including the subventricular zone (SVZ), but also ischemic areas. In vitro, cells isolated from the ischemic areas and the SVZ formed GFP+ neurosphere-like cell clusters that gave rise to various neural lineages including neurons, astrocytes, and oligodendrocytes. However, microarray analysis of these cells and genetic mapping experiments by Nestin-CreERT2 Line4 mice crossed with yellow fluorescent protein (YFP) reporter mice (Nestin promoter-driven YFP-expressing mice) indicated that cells with NSPC activities in the ischemic areas and the SVZ had different characteristics and origins. These results show that the expression patterns and fate of GFP+ cells with NSPC activities can be precisely investigated over a long period in Nestin-GFP mice (CB-17 background), which is not necessarily possible with Nestin-GFP mice (C57BL/6 background). Thus, Nestin-GFP mice (CB-17 background) could become a useful tool with which to investigate the mechanism of neurogenesis via the aforementioned cells under pathological conditions such as following ischemic stroke.
... As the circle of Willis in rats is well-structured, the rats which receive bilateral common carotid artery occlusion (BCCAO) could be developed as experimental models of retinal ischemia [11][12][13]15]. Two common carotid arteries could not be occluded in mice because of a high rate of mouse death (almost 100%) as they may have a lack of posterior communicating arteries in the circle of Willis [17,21,22]. Therefore, bilateral common carotid artery stenosis (BCCAS) has been alternately attempted to induce severe retinal ischemic injuries in mice [14]. ...
Cardiovascular diseases lead to retinal ischemia, one of the leading causes of blindness. Retinal ischemia triggers pathological retinal glial responses and functional deficits. Therefore, maintaining retinal neuronal activities and modulating pathological gliosis may prevent loss of vision. Previously, pemafibrate, a selective peroxisome proliferator-activated receptor alpha modulator, was nominated as a promising drug in retinal ischemia. However, a protective role of pemafibrate remains untouched in cardiovascular diseases-mediated retinal ischemia. Therefore, we aimed to unravel systemic and retinal alterations by treating pemafibrate in a new murine model of retinal ischemia caused by cardiovascular diseases. Adult C57BL/6 mice were orally administered pemafibrate (0.5 mg/kg) for 4 days, followed by unilateral common carotid artery occlusion (UCCAO). After UCCAO, pemafibrate was continuously supplied to mice until the end of experiments. Retinal function (a-and b-waves and the oscillatory potentials) was measured using electroretinography on day 5 and 12 after UCCAO. Moreover, the retina, liver, and serum were subjected to qPCR, immunohistochemistry, or ELISA analysis. We found that pemafibrate enhanced liver function, elevated serum levels of fibroblast growth factor 21 (FGF21), one of the neuroprotective molecules in the eye, and protected against UCCAO-induced retinal dysfunction, observed with modulation of retinal gliosis and preservation of oscillatory potentials. Our current data suggest a promising pemafibrate therapy for the suppression of retinal dysfunction in cardiovascular diseases.
Keywords: common carotid artery occlusion; electroretinography; fibroblast growth factor 21; pemafibrate; peroxisome proliferator-activated receptor alpha; retinal ischemia
... They are easily affected by neurological de cits and brain damage, and the carotid artery of the carotid artery is simply and brie y occluded (Berry K et al. 1975), even as short as 5 minutes. Compared with other strains of mice, C57BL/6 mice undergoing BCCAo surgery showed reliable ischemic neurological symptoms, and had highly reproducible histological damage in the hippocampus and papillae (Yang G et al. 1997). ...
Background: Nerve cell apoptosis is an important pathological mechanism of vascular dementia (VaD). There are evidences that Electroacupuncture (EA) can reduce cognitive impairment in VaD patients and protect nerve cells. However, the mechanism remains unknown. JNK signaling pathway play an important role in apoptosis. And JNK induces cell apoptosis in two main ways, namely JNK transcription-dependent pathway and JNK transcription-independent route. We focus on whether EA can inhibit apoptosis and alleviate cognitive impairment by regulating JNK signaling pathway.
Method: In this study, Bilateral common carotid artery clipping (BCCAo) was used as a method to establish VaD mouse model. Longa scoring method and Morris water maze were used to evaluate whether EA could improve the behavioral score of VaD mice. The neuroprotective effects of EA were evaluated by the Hematoxylin and Eosin (HE) staining, TUNEL and Flow Cytometry (FCM). Western blot and real-time PCR were used to detect the expression of JNK-3, AP-1, P53, Bcl -2, Bax, and Caspase-3.
Results: Our findings indicated that EA could improve the behavioral scores of VaD mice. VaD mice treated with EA reduced hippocampal neuronal apoptosis. In addition, EA could reduce the expression of JNK-3, AP-1, P53, Bax and Caspase-3 proteins and mRNA in the hippocampus of VaD mice, and increase the expression of Bcl-2.
Conclusion: Our findings suggested that the mechanism of action of EA to treat VaD may be related to its regulation of JNK transcription-dependent pathway and JNK transcription-independent route.
... Bilateral common carotid artery occlusion to induce ocular ischemia has been applied to rats, in that the circle of Willis in rats is well-organized [9][10][11][12][13]17]. However, several studies reported that bilateral common carotid artery occlusion could not be applicable to mice with a high rate of death (almost 100%) because of a lack of posterior communicating arteries in the circle of Willis in mice [14,18,19]. Hence, unilateral common carotid artery occlusion (UCCAO) has been applied to mice for understanding pathological mechanisms for ocular ischemia [14,20]. ...
Ocular ischemia is a common cause of blindness and plays a detrimental role in various diseases such as diabetic retinopathy, occlusion of central retinal arteries, and ocular ischemic syndrome. Abnormalities of neuronal activities in the eye occur under ocular ischemic conditions. Therefore, protecting their activities may prevent vision loss. Previously, peroxisome proliferator-activated receptor alpha (PPARα) agonists were suggested as promising drugs in ocular ischemia. However, the potential therapeutic roles of PPARα agonists in ocular ischemia are still unknown. Thus, we attempted to unravel systemic and ocular changes by treatment of fenofibrate, a well-known PPARα agonist, in a new murine model of ocular ischemia. Adult mice were orally administered fenofibrate (60 mg/kg) for 4 days once a day, followed by induction of ocular ischemia by unilateral common carotid artery occlusion (UCCAO). After UCCAO, fenofibrate was continuously supplied to mice once every 2 days during the experiment period. Electroretinography was performed to measure retinal functional changes. Furthermore, samples from the retina, liver, and blood were subjected to qPCR, Western blot, or ELISA analysis. We found that fenofibrate boosted liver function, increased serum levels of fibroblast growth factor 21 (FGF21), one of the neuroprotective molecules in the central nervous system, and protected against UCCAO-induced retinal dysfunction. Our current data suggest a promising fenofibrate therapy in ischemic retinopathies.
... This series of experiments was designed to examine the role of hippocampal neurogenesis in cognitive recovery following hippocampal damage. Global ischemia models, such as bilateral common carotid artery occlusion, reportedly result in a bilateral loss of CA1 hippocampal neurons in C57BL/6 mice due to an abnormality in that strain's cerebrovasculature, but produce less consistent results in other mouse strains [29][30][31][32][33] . Furthermore, recovery treatments require longevity to be relevant 22 . ...
Hippocampal atrophy and cognitive decline are common sequelae of many neurodegenerative disorders, including stroke. To determine whether cognitive decline can be ameliorated by exercise-induced neurogenesis, C57BL/6 mice in which a unilateral hippocampal injury had been induced by injecting the vasoconstrictor endothelin-1 into their right hippocampus, were run voluntarily for 21 days on a running-wheel. We found the severe deficits in spatial learning, as detected by active place-avoidance task, following injury were almost completely restored in animals that ran whereas those that did not run showed no improvement. We show the increase in neurogenesis found in both the injured and contralateral hippocampi following running was responsible for the restoration of learning since bilateral ablation of newborn doublecortin (DCX)-positive neurons abrogated the cognitive improvement, whereas unilateral ablations of DCX-positive neurons did not prevent recovery, demonstrating that elevated neurogenesis in either the damaged or intact hippocampus is sufficient to reverse hippocampal injury-induced deficits.
... After MCAo-stroke we found a 5 × increase in apoptotic activity within the ischaemic lesion. Increased caspase-3 activity is predominately localised to the CA3 region of the hippocampus and striatum 24 h after global ischaemia induced in the BCCAo model 9,30 . We observed that overall, after DCAL-stroke apoptosis in the ischaemic lesion was comparatively less than that of the MCAo cohorts. ...
Stroke is caused by obstructed blood flow (ischaemia) or unrestricted bleeding in the brain (haemorrhage). Global brain ischaemia occurs after restricted cerebral blood flow e.g. during cardiac arrest. Following ischaemic injury, restoration of blood flow causes ischaemia–reperfusion (I/R) injury which worsens outcome. Secondary injury mechanisms after any stroke are similar, and encompass inflammation, endothelial dysfunction, blood–brain barrier (BBB) damage and apoptosis. We developed a new model of transient global forebrain I/R injury (dual carotid artery ligation; DCAL) and compared the manifestations of this injury with those in a conventional I/R injury model (middle-cerebral artery occlusion; MCAo) and with intracerebral haemorrhage (ICH; collagenase model). MRI revealed that DCAL produced smaller bilateral lesions predominantly localised to the striatum, whereas MCAo produced larger focal corticostriatal lesions. After global forebrain ischaemia mice had worse overall neurological scores, although quantitative locomotor assessment showed MCAo and ICH had significantly worsened mobility. BBB breakdown was highest in the DCAL model while apoptotic activity was highest after ICH. VCAM-1 upregulation was specific to ischaemic models only. Differential transcriptional upregulation of pro-inflammatory chemokines and cytokines and TLRs was seen in the three models. Our findings offer a unique insight into the similarities and differences in how biological processes are regulated after different types of stroke. They also establish a platform for analysis of therapies such as endothelial protective and anti-inflammatory agents that can be applied to all types of stroke.
... As mice lack fully developed posterior communicating arteries (PCAs) in the circle of Willis, BCCAO severely interrupts blood supply to the brain, eventually leading to death (Venkat et al., 2015; Abbreviations: BCCAO, bilateral common carotid artery occlusion; BCCAS, bilateral common carotid artery stenosis; CCA, common carotid artery; ECA, external carotid artery; GCL, ganglion cell layer; HIF, hypoxia-inducible factor; ICA, internal carotid artery; INL, inner nuclear layer; IPL, inner plexiform layer; OIS, ocular ischemic syndrome; ONL, outer nuclear layer; UCCAO, unilateral common carotid artery occlusion. Wellons et al., 2000;Yang et al., 1997), while the brain of rats can be supplied from vertebral arteries via the circle of Willis even after BCCAO. Although some researchers have utilized mice using the bilateral CCA stenosis (BCCAS) technique to study retinal ischemia (Crespo-Garcia et al., 2018), individual variation and chance of severe brain damages could not be excluded. ...
Retina, one of the highest oxygen demanding tissues, is vulnerable to vascular insufficiencies, and various ocular vascular disorders can cause chronic retinal ischemia. To investigate the pathophysiology, rodent models developed by bilateral common carotid artery occlusion (BCCAO) have been utilized. However, mice lack posterior communicating arteries in the circle of Willis and cannot endure the brain ischemia induced by the bilateral occlusion. A mouse model to better reflect the localized ischemic stress in the retina without affecting the brain is still needed. Here, we established a mouse model of ischemic injury by permanent unilateral common carotid artery occlusion (UCCAO). Adult male mice were subjected to UCCAO, and changes in the ipsilateral retina were examined in comparison with the contralateral retina. Delayed perfusion was observed in the ipsilateral retina right after the occlusion and was not recovered later on. Common features of retinal ischemia were observed: Hypoxia-inducible factor (HIF) stabilization; upregulation of hypoxia-responsive genes; altered levels of cytokines and chemokines. Activation of astrocytes and Müller cells in the inner retina was detected at day 2, and thinning of the inner retinal layer became significant at week 10. Together, our model can simulate retinal ischemia with morphological and molecular changes. It can be utilized to investigate pathophysiology of ischemic retinopathies.
... It was not surprise because of B57BL/6 was the most susceptible to global cerebral ischemia rather than ICR or other mouse strains, based on neurological signs, histological fi ndings, cortical microcirculatory and perfusion patterns (Yang et al. 1997).There were reports about the differences of pathophysiology and behavioral outcome among mouse strains (Adams et al. 2002, Brosnan-Watters et al. 2000. Not only strain difference, sex also concern especially in the cognitive behavioral tests (Ge et al. 2013). ...
... Anatomical variations between mouse strains, particularly vascular differences, are often proposed to be the cause for differences in experimental outcome in stroke and hypoxic-ischemic encephalopathy [28]. An adult mouse study, for example, showed the C57Bl/6 strain to have the poorest development of the Circle of Willis of the 7 strains studied (not including CD1), as well as the highest mortality and greatest susceptibility to injury after bilateral common carotid artery occlusion [29]. ...
Neonatal hypoxic-ischemic brain injury is commonly studied by means of the Vannucci procedure in mice or rats (unilateral common carotid artery occlusion followed by hypoxia). Previously, we modified the postnatal day 7 (P7) rat procedure for use in mice, and later demonstrated that genetic strain strongly influences the degree of brain injury in the P7 mouse model of hypoxia-ischemia (HI). Recently, the P9 or P10 mouse brain was recognized as the developmental equivalent of a term neonatal human brain, rather than P7. Consequently, the Vannucci procedure has again been modified, and a commonly used protocol employs 10% oxygen for 50 min in C57Bl/6 mice. Strain differences have yet to be described for the P9/P10 mouse model. In order to determine if the strain differences we previously reported in the P7 mouse model are present in the P9 model, we compared 2 commonly used strains, CD1 and C57Bl/6J, in both the P7 (carotid ligation [in this case, right] followed by exposure to 8% oxygen for 30 min) and P9 (carotid ligation [in this case left] followed by exposure to 10% oxygen) models of HI. Experiments using the P7 model were performed in 2001-2012 and those using the P9 model were performed in 2012-2016. Five to seven days after the HI procedure, mice were perfused with 4% paraformaldehyde, their brains were sectioned on a Vibratome (50 μm) and alternate sections were stained with Perl's iron stain or cresyl violet. Brain sections were examined microscopically and scored for the degree of injury. Since brains in the P7 group had been scored previously with a slightly different system, they were reanalyzed using our current scoring system which scores injury in 11 regions: the anterior, middle, and posterior cortex; the anterior, middle, and posterior striatum; CA1, CA2, CA3, and the dentate gyrus of the hippocampus and thalamus, on a scale from 0 (none) to 3 (cystic infarct) for a total score of 0-33. Brains in the P9 group were scored with the same system. Given the same insult, the P7 CD1 mice had greater injury than the C57Bl/6J mice, which agrees with our previous findings. The P9 CD1 mice also had greater injury than the C57Bl/6J mice. This study confirms that CD1 mice are more susceptible to injury than C57Bl/6J mice and that strain selection is important when using mouse models of HI.
... The aim of this study was to assess whether this AUR/NGIN-rich test juice would have a neuroprotective effect against the damage caused to the mouse brain by transient cerebral global ischemia. As a model animal of transient cerebral global ischemia, C57BL/6 strain mice were subjected to bilateral common carotid occlusion (2-vessel occlusion: 2VO), because this strain is most susceptible to cerebral ischemia following 2VO among seven common mouse strains (C57BL/6, ICR, BALB/c, C3H, CBA, ddY and DBA/2) (19). ...
Cerebral ischemia/reperfusion leads to delayed neuronal cell death, resulting in brain damage. Auraptene (AUR) and naringin (NGIN), which exert neuroprotective effects in ischemic brain, are abundant in the peel of Citrus kawachiensis. Although parts of AUR/NGIN are transited from the peel to the juice during the squeezing of this fruit, these amounts in juice might be too low to exert effects. We thus prepared the AUR/NGIN-rich fruit juice of C. kawachiensis by addition of peel paste to the raw juice. The present study revealed that orally administration of the dried powder of this AUR/NGIN-rich fruit juice (2.5 g/kg/d) for 7 d to ischemic mice significantly suppressed the ischemia-induced neuronal cell death in the hippocampus, which was coincidently with the reduction of hyperactivation of microglia and astrocytes. These results suggest that AUR/NGIN-rich juice of C. kawachiensis may possess therapeutic potential for the prevention of neurodegenerative diseases via inhibition of inflammatory processes.
... In this study, we comprehensively evaluated recovery of learning and memory functions in the global cerebral ischemic model, in which it was found that cognitive function was deficient due to neuronal damage, degeneration and apoptosis in the acute stage after reperfusion. Evidence suggested that C57BL/6 mice are particularly susceptible to neuronal damage in the CA1 region of the hippocampus after transient global ischemia (Yang et al., 1997). The present results also showed that TAT-Ngn2 treatment improved the recovery of cognitive function and alleviated neuronal damage, degeneration and apoptosis in vivo and in vitro. ...
Neurogenin-2 (Ngn2) is a basic helix-loop-helix (bHLH) transcription factor that contributes to the identification and specification of neuronal fate during neurogenesis. In our previous study, we found that Ngn2 plays an important role in alleviating neuronal apoptosis, which may be viewed as an attractive candidate target for the treatment of cerebral ischemia. However, novel strategies require an understanding of the function and mechanism of Ngn2 in mature hippocampal neurons after global cerebral ischemic injury. Here, we found that the expression of Ngn2 decreased in the hippocampus after global cerebral ischemic injury in mice and in primary hippocampal neurons after oxygen glucose deprivation (OGD) injury. Then, transactivator of transcription (TAT)-Ngn2, which was constructed by fusing a TAT domain to Ngn2, was effectively transported and incorporated into hippocampal neurons after intraperitoneal (i.p.) injection and enhanced cognitive functional recovery in the acute stage after reperfusion. Furthermore, TAT-Ngn2 alleviated hippocampal neuronal damage and apoptosis, and inhibited the cytochrome C (CytC) leak from the mitochondria to the cytoplasm through regulating the expression levels of brain-derived neurotrophic factor (BDNF), phosphorylation tropomyosin-related kinase B (pTrkB), Bcl-2, Bax and cleaved caspase-3 after reperfusion injury in vivo and in vitro. These findings suggest that the downregulation of Ngn2 expression may have an important role in triggering brain injury after ischemic stroke and that the neuroprotection of TAT-Ngn2 against stroke might involve the modulation of BDNF-TrkB signaling that regulates caspase-dependent and mitochondrial apoptotic pathways, which may be an attractive therapeutic strategy for cerebral ischemic injury.
... Since the strain differences in arterial collaterals and sensitivity to excitotoxic cell death, mice exhibit profound inter-strain differences in infarct volumes after MCAO (Carmichael, 2005), which is common focal stroke model of intra-arterial suture occlusion of the MCA. Several studies confirmed that C57BL/6 mice showed significantly larger infarct volumes than the Sv129 strain (Connolly et al., 1996;Maeda et al., 1999), and in global ischemia, C57BL/6 is more sensitive than BALB/c and other strains (Yang et al., 1997). However, in distal MCAO (see below), cortical infarcts in BALB/c mice were 3-fold larger than those in 129X1/SvJ and C57BL/6J mice (Majid et al., 2000). ...
Stroke is one of the leading causes of death worldwide, and the majority of cerebral stroke is caused by occlusion of cerebral circulation, which eventually leads to brain infarction. Although stroke occurs mainly in the aged population, most animal models for experimental stroke in vivo almost universally rely on young-adult rodents for the evaluation of neuropathological, neurological, or behavioral outcomes after stroke due to their greater availability, lower cost, and fewer health problems. However, it is well established that aged animals differ from young animals in physiology, neurochemistry, and behavior. Stroke-induced changes are more pronounced with advancing age. Therefore, the overlooked role of age in animal models of stroke could impact on data quality and hinder the translation of rodent models to humans. In addition to aging, other factors also influence the performance after ischemic stroke. In this article, we summarize the differences between young and aged animals, the impact of age, sex and animal strains on performance and outcome in the animal models of stroke and emphasize age as a key factor in preclinical stroke studies in animal models of stroke.
... In animal studies of traumatic CNS injury worldwide, no effort was made to find a link between neuronal recovery and the resistance or susceptibility of a particular strain to autoimmune disease. Studies have attempted to link the outcome of neuronal trauma to genetic (Friedman et al., 1999;Mattson et al., 2000) or anatomic (Yang et al., 1997) differences between strains, but no attention was directed to the possibility that the response to trauma is controlled by the immune system. Indeed, the traditional view was that if CNS insult evokes any immune response at all, it could only be detrimental for neuronal survival. ...
Injury to the CNS is often followed by a spread of damage (secondary degeneration), resulting in neuronal losses that are substantially greater than might have been predicted from the severity of the primary insult. Studies in our laboratory have shown that injured CNS neurons can benefit from active or passive immunization with CNS myelin-associated antigens. The fact that autoimmune T-cells can be both beneficial and destructive, taken together with the established phenomenon of genetic predisposition to autoimmune diseases, raises the question: will genetic predisposition to autoimmune diseases affect the outcome of traumatic insult to the CNS? Here we show that the survival rate of retinal ganglion cells in adult mice or rats after crush injury of the optic nerve or intravitreal injection of a toxic dosage of glutamate is up to twofold higher in strains that are resistant to the CNS autoimmune disease experimental autoimmune encephalomyelitis (EAE) than in susceptible strains. The difference was found to be attributed, at least in part, to a beneficial T-cell response that was spontaneously evoked after CNS insult in the resistant but not in the susceptible strains. In animals of EAE-resistant but not of EAE-susceptible strains devoid of mature T-cells (as a result of having undergone thymectomy at birth), the numbers of surviving neurons after optic nerve injury were significantly lower (by 60%) than in the corresponding normal animals. Moreover, the rate of retinal ganglion cell survival was higher when the optic nerve injury was preceded by an unrelated CNS (spinal cord) injury in the resistant strains but not in the susceptible strains. It thus seems that, in normal animals of EAE-resistant strains (but not of susceptible strains), the injury evokes an endogenous protective response that is T-cell dependent. These findings imply that a protective T-cell-dependent response and resistance to autoimmune disease are regulated by a common mechanism. The results of this study compel us to modify our understanding of autoimmunity and autoimmune diseases, as well as the role of autoimmunity in non-autoimmune CNS disorders. They also obviously have far-reaching clinical implications in terms of prognosis and individual therapy.
... The pathogenesis of the two forms of ischemia do not overlap. In global ischemia, although cardiovascular diseases leading to cardiac arrest have been cited as the primary cause [5][6][7], there have been reports of bi-lateral occlusion of the common carotid artery in the mouse model as a potential causality [8]. Contrastingly, etiologies of focal ischemia have been *Address correspondence to this author at the Department of Anatomy, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Center, Jalan Yaacob Latif, Bandar Tun Razak 56000 Kuala Lumpur, Malaysia; Tel: +603-91458634; Fax: +603-91458607; E-mail: mfairuzy@ukm.edu.my ...
Brain ischemia is among the leading cause of death with majority of the cases are associated with ischemic strokes. It can occur in two form of either focal or global ischemia. Neurodegenerative disorder such as Alzheimer and Parkinson diseases is also on the rise worldwide. These disorders have common similarities; i.e. they all affecting the central nervous system with debilitating effect to the patient. In this review, we look into the promising role of flavonoids, a natural bioactive compound found abundant in vegetables, fruits and traditional herbs. Treatment with flavonoids such as curcumin, lycopene, ginsenoside, vitexin and baicalin have shown promising neuroprotective effects against ischemic-induced injury. Besides anticancer, antioxidant and immunomodulation properties, flavonoid also exerts neuroprotective effects by increases neuronal viability, increases tissue perfusion and cerebral blood flow and reduce ischemic-related apoptosis. In addition, flavonoid also exerts anti-amyloidogenic effect and reduces loss of dopaminergic neurons in the brain. These results suggesting flavonoids might be able to serve as a potential therapeutic agent in brain disorders.
... Several studies (Bottiger et al 1999;Olsson et al 2003;Yang et al 1997;Yonekura et al 2004) have shown that with an inspired gas mixture of 30% O2 and 70% N2O, the pre-ischemia levels of partial oxygen pressure (PaO2), partial carbon dioxide pressure (PaCO2), and pH varied from 93.4 ± 21.1 to 208 ± 45 mmHg, from 28.1 ± 4.6 to 40 ± 5 mmHg, and from 7.12 ± 0.04 to 7.39 ± 0.08, respectively. One likely reason for these large variations may be that different tidal volume and respiratory rates were used throughout these studies. ...
... The resulting reductions in blood flow are severe, whereby cortical blood flow drops by over 70% in the days immediately following surgery, recovering to a 40% reduction in 1 month [29]. In C57Bl/6J mice, complete occlusion of both carotid arteries leads to death due to poor collateral flow through the Circle of Willis, and as an alternative the carotid arteries are temporarily occluded for durations lasting no more than 30 min [30]. This approach leads to transient global ischaemia with blood flow reduced by 80-90% [31,32]. ...
Increasing evidence suggests that vascular risk factors contribute to neurodegeneration, cognitive impairment and dementia. While there is considerable overlap between features of vascular cognitive impairment and dementia (VCID) and Alzheimer's disease (AD), it appears that cerebral hypoperfusion is the common underlying pathophysiological mechanism which is a major contributor to cognitive decline and degenerative processes leading to dementia. Sustained cerebral hypoperfusion is suggested to be the cause of white matter attenuation, a key feature common to both AD and dementia associated with cerebral small vessel disease (SVD). White matter changes increase the risk for stroke, dementia and disability. A major gap has been the lack of mechanistic insights into the evolution and progress of VCID. However, this gap is closing with the recent refinement of rodent models which replicate chronic cerebral hypoperfusion. In this review, we discuss the relevance and advantages of these models in elucidating the pathogenesis of VCID and explore the interplay between hypoperfusion and the deposition of amyloid β (Aβ) protein, as it relates to AD. We use examples of our recent investigations to illustrate the utility of the model in preclinical testing of candidate drugs and lifestyle factors. We propose that the use of such models is necessary for tackling the urgently needed translational gap from preclinical models to clinical treatments.
Basic relevant information on methodologies used in neurological disease models can be extremely hard to find. Originally published in 2006, this important reference work contains 30 chapters from over 60 internationally recognized scientists and covers every major methodology and disease model used in neuroscience research. Divided into two major sections, the first deals with general methodologies in neuroscience research covering topics from animal welfare and ethical issues to surgical procedures, post-operative care and behavioral testing. Section two covers every major disease model including traumatic brain injury, ischemia and stroke, to Parkinson's, motor neurone disease, epilepsy and sleep disorders. Delivering critical methodological information and describing small animal models for almost all major neurological diseases, this book forms an essential reference for anyone working in neuroscience, from beginning students to experienced researchers and medical professionals.
Basic relevant information on methodologies used in neurological disease models can be extremely hard to find. Originally published in 2006, this important reference work contains 30 chapters from over 60 internationally recognized scientists and covers every major methodology and disease model used in neuroscience research. Divided into two major sections, the first deals with general methodologies in neuroscience research covering topics from animal welfare and ethical issues to surgical procedures, post-operative care and behavioral testing. Section two covers every major disease model including traumatic brain injury, ischemia and stroke, to Parkinson's, motor neurone disease, epilepsy and sleep disorders. Delivering critical methodological information and describing small animal models for almost all major neurological diseases, this book forms an essential reference for anyone working in neuroscience, from beginning students to experienced researchers and medical professionals.
This study aimed to compare the effects of bilateral common carotid artery occlusion (BCCAO) and bilateral carotid artery stenosis (BCAS) on brain function and structure in rodents. Adult male Sprague-Dawley rats and C57BL/6 mice were randomly assigned to either the BCCAO or sham group and the BCAS or sham group, respectively. Neurobehavioral assessments were conducted using various tests, and histological examination was performed to evaluate brain structure. Both surgeries induced white matter damage, with BCCAO mainly affecting the hippocampus and BCAS affecting the blood flow. BCCAO rats exhibited anxiety-like behaviors and cognitive impairment at week 8, while BCAS mice showed significant cognitive deficits at week 4. Both surgeries caused cellular responses to white matter damage, with increased astrocytes and microglial/macrophagemarkers observed in the corpus callosum region. Physical growth and development were not significantly impacted, and there was no difference in bilateral cerebral blood flow between the surgery and sham groups. In conclusion, both models induced significant cognitive impairment and white matter damage. BCCAO induced anxiety-like behaviors at a later stage compared to BCAS, which could help researchers study the underlying mechanisms of chronic cerebral ischemia and drug evaluation.
Basic relevant information on methodologies used in neurological disease models can be extremely hard to find. Originally published in 2006, this important reference work contains 30 chapters from over 60 internationally recognized scientists and covers every major methodology and disease model used in neuroscience research. Divided into two major sections, the first deals with general methodologies in neuroscience research covering topics from animal welfare and ethical issues to surgical procedures, post-operative care and behavioral testing. Section two covers every major disease model including traumatic brain injury, ischemia and stroke, to Parkinson's, motor neurone disease, epilepsy and sleep disorders. Delivering critical methodological information and describing small animal models for almost all major neurological diseases, this book forms an essential reference for anyone working in neuroscience, from beginning students to experienced researchers and medical professionals.
Basic relevant information on methodologies used in neurological disease models can be extremely hard to find. Originally published in 2006, this important reference work contains 30 chapters from over 60 internationally recognized scientists and covers every major methodology and disease model used in neuroscience research. Divided into two major sections, the first deals with general methodologies in neuroscience research covering topics from animal welfare and ethical issues to surgical procedures, post-operative care and behavioral testing. Section two covers every major disease model including traumatic brain injury, ischemia and stroke, to Parkinson's, motor neurone disease, epilepsy and sleep disorders. Delivering critical methodological information and describing small animal models for almost all major neurological diseases, this book forms an essential reference for anyone working in neuroscience, from beginning students to experienced researchers and medical professionals.
Basic relevant information on methodologies used in neurological disease models can be extremely hard to find. Originally published in 2006, this important reference work contains 30 chapters from over 60 internationally recognized scientists and covers every major methodology and disease model used in neuroscience research. Divided into two major sections, the first deals with general methodologies in neuroscience research covering topics from animal welfare and ethical issues to surgical procedures, post-operative care and behavioral testing. Section two covers every major disease model including traumatic brain injury, ischemia and stroke, to Parkinson's, motor neurone disease, epilepsy and sleep disorders. Delivering critical methodological information and describing small animal models for almost all major neurological diseases, this book forms an essential reference for anyone working in neuroscience, from beginning students to experienced researchers and medical professionals.
Basic relevant information on methodologies used in neurological disease models can be extremely hard to find. Originally published in 2006, this important reference work contains 30 chapters from over 60 internationally recognized scientists and covers every major methodology and disease model used in neuroscience research. Divided into two major sections, the first deals with general methodologies in neuroscience research covering topics from animal welfare and ethical issues to surgical procedures, post-operative care and behavioral testing. Section two covers every major disease model including traumatic brain injury, ischemia and stroke, to Parkinson's, motor neurone disease, epilepsy and sleep disorders. Delivering critical methodological information and describing small animal models for almost all major neurological diseases, this book forms an essential reference for anyone working in neuroscience, from beginning students to experienced researchers and medical professionals.
As well as their ion transportation function, the voltage-dependent potassium channels could act as the cell signal inducer in a variety of pathogenic processes. However, their roles in neurogenesis after stroke insults have not been clearly illustrated. In our preliminary study, the expressions of voltage-dependent potassium channels Kv4.2 was significantly decreased after stroke in cortex, striatum and hippocampus by real-time quantitative PCR assay. To underlie the neuroprotection of Kv4.2 in stroke rehabilitation, recombinant plasmids encoding the cDNAs of mouse Kv4.2 was constructed. Behavioral tests showed that the increased Kv4.2 could be beneficial to the recovery of the sensory, the motor functions and the cognitive deficits after stroke. Temozolomide (TMZ), an inhibitor of neurogenesis, could partially abolish the mentioned protections of Kv4.2. The immunocytochemical staining showed that Kv4.2 could promote the proliferations of neural stem cells and induce the neural stem cells to differentiate into neurons in vitro and in vivo. And Kv4.2 could up-regulate the expressions of ERK1/2, p-ERK1/2, p-STAT3, NGF, p-TrkA, and BDNF, CAMKII and the concentration of intracellular Ca2⁺. Namely, we concluded that Kv4.2 promoted neurogenesis through ERK1/2/STAT3, NGF/TrkA, Ca²⁺/CAMKII signal pathways and rescued the ischemic impairments. Kv4.2 might be a potential drug target for ischemic stroke intervention.
Inhibition of phosphodiesterase 4 (PDE4) is a promising pharmacological strategy for the treatment of cerebral ischemic conditions. To increase the relevance and increase the translational value of preclinical studies, it is important to conduct experiments using different animal species and strains, different animal models, and to evaluate long-term functional outcomes after cerebral ischemia. In the present study, the effects of the selective PDE4 inhibitor roflumilast were evaluated in vivo and in vitro. Balb/c mice were subjected to bilateral common carotid artery occlusion (BCCAO) and tested during 21 days in multiple behavioral tasks to investigate the long-term effects of roflumilast on functional recovery. The effects of roflumilast were also investigated on hippocampal cell loss, white matter injury, and expression of neuroinflammatory markers. Roflumilast prevented cognitive and emotional deficits induced by BCCAO in mice. Roflumilast also prevented neurodegeneration and reduced the white matter damage in the brain of ischemic animals. Besides, roflumilast decreased Iba-1 (microglia marker) levels and increased Arginase-1 (Arg-1; microglia M2 phenotype marker) levels in the hippocampus of these mice. Likewise, roflumilast suppressed inducible nitric oxide synthase (microglia M1 phenotype marker) expression and increased Arg-1 levels in a primary mouse microglia culture. These findings support evidence that PDE4 inhibition by roflumilast might be beneficial in cerebral ischemic conditions. The neuroprotective effects of roflumilast appear to be mediated by a decrease in neuroinflammation.
Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. To investigate pathological mechanisms of retinal ischemia, relevant experimental models need to be developed. Anatomically, a main retinal blood supplying vessel is the ophthalmic artery (OpA) and OpA originates from the internal carotid artery of the common carotid artery (CCA). Thus, disruption of CCA could effectively cause retinal ischemia. Here, we established a mouse model of retinal ischemia by transient bilateral common carotid artery occlusion (tBCCAO) to tie the right CCA with 6-0 silk sutures and to occlude the left CCA transiently for 2 seconds via a clamp, and showed that tBCCAO could induce acute retinal ischemia leading to retinal dysfunction. The current method reduces reliance on surgical instruments by only using surgical needles and a clamp, shortens occlusion time to minimize unexpected animal death, which is often seen in mouse models of middle cerebral artery occlusion, and maintains reproducibility of common retinal ischemic findings. The model can be utilized to investigate the pathophysiology of ischemic retinopathies in mice and further can be used for in vivo drug screening.
Background and purpose:
Intracranial aneurysm formation and rupture risk are, in part, determined by genetic factors and sex. To examine their role, we compared 3 mouse strains commonly used in cerebrovascular studies in a model of intracranial aneurysm formation and rupture.
Methods:
Intracranial aneurysms were induced in male CD1 (Crl:CD1[ICR]), male and female C57 (C57BL/6NCrl), and male 129Sv (129S2/SvPasCrl or 129S1/SvImJ) mice by stereotaxic injection of elastase at the skull base, combined with systemic deoxycorticosterone acetate-salt hypertension. Neurological deficits and mortality were recorded. Aneurysms and subarachnoid hemorrhage grades were quantified postmortem, either after spontaneous mortality or at 7 to 21 days if the animals survived. In separate cohorts, we examined proinflammatory mediators by quantitative reverse transcriptase-polymerase chain reaction, arterial blood pressure via the femoral artery, and the circle of Willis by intravascular latex casting.
Results:
We found striking differences in aneurysm formation, rupture, and postrupture survival rates among the groups. 129Sv mice showed the highest rates of aneurysm rupture (80%), followed by C57 female (36%), C57 male (27%), and CD1 (21%). The risk of aneurysm rupture and the presence of unruptured aneurysms significantly differed among all 3 strains, as well as between male and female C57. The same hierarchy was observed upon Kaplan-Meier analysis of both overall survival and deficit-free survival. Subarachnoid hemorrhage grades were also more severe in 129Sv. CD1 mice showed the highest resistance to aneurysm rupture and the mildest outcomes. Higher mean blood pressures and the major phenotypic difference in the circle of Willis anatomy in 129Sv provided an explanation for the higher incidence of and more severe aneurysm ruptures. TNFα (tumor necrosis factor-alpha), IL-1β (interleukin-1-beta), and CCL2 (chemokine C-C motif ligand 2) expressions did not differ among the groups.
Conclusions:
The outcome of elastase-induced intracranial aneurysm formation and rupture in mice depends on genetic background and shows sexual dimorphism.
Background: The molecular mechanisms of vascular cognitive impairment (VCI) are diverse and still in puzzle. VCI could be attributed to chronic cerebral hypoperfusion (CCH). CCH may cause a cascade of reactions involved in ischemia and neuro-inflammation which plays important roles in the pathophysiology of VCI. High-mobility group box protein 1 (HMGB1) is a non-histone protein that serves as a damage-associated molecular signal leading to cascades of inflammation. Increased level of HMGB1 has been established in the acute phase of CCH. However, the role of HMGB1 at the chronic phase of CCH remains elucidated. Methods: We performed modified bilateral common carotid artery occlusion (BCCAO) in C57BL/6 mice to induce CCH. We examined the cerebral blood flow (CBF) reduction by laser doppler flowmetry, the protein expression of HMGB1 and its pro-inflammatory cytokines (TNF-a, IL-1b, and IL-6) by western blotting and immunohistochemistry. The brain pathology was assessed by 7T-animal MRI and amyloid-b accumulation was assessed by amyloid-PET scanning. We further evaluated the effect of HMGB1 suppression by injecting CRISPR/Cas9 knock-out plasmid intra-hippocampus bilaterally. Results: There were reduction of CBF up to 50% which persisted three months after CCH. The modified-BCCAO animals developed significant cognitive decline. The 7T-MRI image showed hippocampal atrophy, although the amyloid-PET showed no significant amyloid-beta accumulation. Increased protein levels of HMGB1, TNF-a and IL-1b were found three months after BCCAO. HMGB1 suppression by CRISPR/Cas9 knock-out plasmid restored the CBF, IL-1B, TNF-alpha, IL-6, and attenuated hippocampal atrophy and cognitive decline. Conclusion: HMGB1 plays a pivotal role in the pathophysiology of the animal model of CCH and it might be a candidate as therapeutic targets of VCI.
The pathophysiology of vascular cognitive impairment (VCI) is associated with chronic cerebral hypoperfusion (CCH). Increased high-mobility group box protein 1 (HMGB1), a nonhistone protein involved in injury and inflammation, has been established in the acute phase of CCH. However, the role of HMGB1 in the chronic phase of CCH remains unclear. We developed a novel animal model of CCH with a modified bilateral common carotid artery occlusion (BCCAO) in C57BL/6 mice. Cerebral blood flow (CBF) reduction, the expression of HMGB1 and its proinflammatory cytokines (tumor necrosis factor-alpha [TNF-α], interleukin [IL]-1β, and IL-6), and brain pathology were assessed. Furthermore, we evaluated the effect of HMGB1 suppression through bilateral intrahippocampus injection with the CRISPR/Cas9 knockout plasmid. Three months after CCH induction, CBF decreased to 30–50% with significant cognitive decline in BCCAO mice. The 7T-aMRI showed hippocampal atrophy, but amyloid positron imaging tomography showed nonsignificant amyloid-beta accumulation. Increased levels of HMGB1, TNF-α, IL-1β, and IL-6 were observed 3 months after BCCAO. HMGB1 suppression with CRISPR/Cas9 knockout plasmid restored TNF-α, IL-1β, and IL-6 and attenuated hippocampal atrophy and cognitive decline. We believe that HMGB1 plays a pivotal role in CCH-induced VCI pathophysiology and can be a potential therapeutic target of VCI.
We aimed to explore the circulating microRNAs biomarkers in the acute stage following cerebral ischemia to earlier warn late-onset post-stroke depression (PSD). A total of 251 consecutive patients with acute ischemic stroke were recruited. They were divided into three groups depending on whether PSD had occurred at 2 weeks or 3 months since stroke: early-onset PSD, late-onset PSD, and non-depressed group. Microarray assay was conducted to identify the different expression profiles of plasma miRNAs. Comprehensive bioinformatics analysis for their integrating putative target genes was performed. The key miRNA was validated in a larger cohort and its function was further studied in ischemic mice brain. We screened three differentially expressed miRNAs in the late-onset PSD individuals, miR-140-5p and miR-221-3p were significantly upregulated while miR-1246 was downregulated. The bioinformatics analysis demonstrated that their predicted target genes were mainly enriched in axon development and Ras signaling pathway. Logistic regression analysis revealed that miR-140-5p was an independent risk factor for late-onset PSD (P = 0.017, OR = 2.313, 95%CI 1.158 to 4.617). The miR-140-5p expression on admission was significantly positively correlated with HDRS scores assessed at 3 months after stroke (P = 0.0007). The predictive value of miR-140-5p for late-onset PSD is 83.3% sensitivity and 72.6% specificity (AUC = 0.8127, P < 0.0001). AAV-mediated overexpression of miR-140-5p decreased the protein level of IL1rap, IL1rapl1, VEGF, and MEGF10 in the ischemic mouse hippocampus and inhibited neurogenesis and capillary density. MiR-140-5p might be involved in the pathogenesis of late-onset PSD and used as a novel early warning biomarker.
Cerebral ischemia is known to be a major cause of death and the later development of Alzheimer's disease and vascular dementia. However, ischemia induced cellular damage that initiates these diseases remain poorly understood. This is primarily due to lack of clinically relevant models that are highly reproducible. Here, we have optimised a murine model of global cerebral ischaemia with multiple markers to determine brain pathology, neurochemistry and correlated memory deficits in these animals. Cerebral ischaemia in mice was induced by bilateral common carotid artery occlusion. Following reperfusion, the mice were either fixed with 4% paraformaldehyde or decapitated under anaesthesia. Brains were processed for Western blotting or immunohistochemistry for glial (GLT1) and vesicular (VGluT1, VGluT2) glutamate transporters and paired helical filament (PHF1) tau. The PHF1 tau is the main component of neurofibrillary tangle, which is the pathological hallmarks of Alzheimer's disease and vascular dementia. The novel object recognition behavioural assay was used to investigate the functional cognitive consequences in these mice. The results show consistent and selective neuronal and glial cell changes in the hippocampus and the cortex together with a significant reduction in GLT1 (***P < 0.001), VGluT1 (**P < 0.01) and VGluT2 (***P < 0.001) expression in the hippocampus in occluded mice as compared to sham-operated animals. These changes are associated with increased PHF1 (***P < 0.0001) protein and a significant impairment of performance (*p < 0.0006, N = 6/group) in the novel object recognition test. This model represents a useful tool for investigating cellular, biochemical and molecular mechanisms of global cerebral ischaemia and may be an ideal preclinical model for vascular dementia.
Memory deficit is the most visible symptom of cerebral ischemia. The hippocampus is sensitive against cerebral ischemia. Oxidative stress and inflammation are involved in the pathological process after cerebral ischemic injury. Paroxetine has anti-oxidative and anti-inflammatory effects. In this study, the effect of paroxetine on memory deficit after cerebral ischemia was investigated. Cerebral ischemia/reperfusion (I/R) injury model was established using the bilateral occlusion of common carotid artery method. Paroxetine (10 mg/kg) was intraperitoneally injected into rats, 24 h before surgery or once a day for 7 days after surgery. Learning and memory were evaluated using the Morris water maze task, then the brain tissue was fixed and hippocampal CA1 pyramidal cells damage was analyzed using the Nissl staining method. In the ischemia group the escape latency time (ELT) and the swimming path length (SPL) were significantly increased and the time spent in target quadrant (TSTQ) was significantly decreased compared with the control group. The ELT and the SPL were significantly shortened and the TSTQ was significantly increased compared with the ischemia group after Pre- or post-ischemic administration of paroxetine. The percentage of viable pyramidal cells in the ischemia group was significantly decreased compared with the control group. The percentage of viable cells was significantly increased following pre-or post-ischemic administration of paroxetine compared with the ischemia group. Memory deficit due to I/R was improved and the percentage of viable cells in CA1 region was increased after administration of paroxetine. Therefore, paroxetine may have a neuroprotective effect against cerebral ischemia. © 2018 by School of Pharmacy Shaheed Beheshti University of Medical Sciences and Health Services.
Sensorimotor recovery after spinal cord injury (SCI) is of utmost importance to injured individuals and will rely on improved understanding of SCI pathology and recovery. Novel transgenic mouse lines facilitate discovery, but must be understood to be effective. The purpose of this study was to characterize the sensory and motor behavior of a common transgenic mouse line (Thy1-GFP-M) before and after SCI. Thy1-GFP-M positive (TG+) mice and their transgene negative littermates (TG-) were acquired from two sources (in-house colony n=32, Jackson Laboratories n=4). C57BL/6J wild-type (WT) mice (Jackson Laboratories n=10) were strain controls. Moderate-severe T9 contusion (SCI) or transection (TX) occurred in TG+ (SCI n=25, TX n=5), TG- (SCI n=5), and WT (SCI n=10) mice. To determine responsiveness to rehabilitation, a cohort of TG+ mice with SCI (n=4) had flat treadmill (TM) training 42-49 days post injury (dpi). To characterize recovery, we performed Basso Mouse Scale, Grid Walk, von Frey Hair, and Plantar Heat testing prior to and out to 42d post SCI. Open field locomotion was significantly better in Thy1 SCI groups (TG+ and TG-) compared to WT by 7dpi (p<0.01) and was maintained through 42 dpi (p<0.01). These unexpected locomotor gains were not apparent during grid walking, indicating severe impairment of precise motor control. Thy1 derived mice were hypersensitive to mechanical stimuli at baseline (p<0.05). After SCI, mechanical hyposensitivity emerged in Thy1 derived groups (p<0.001), while thermal hyperalgesia occurred in all groups (p<0.001). Importantly, consistent findings across TG+ and TG- groups suggest that the effects are mediated by the genetic background rather than transgene manipulation itself. Surprisingly, TM training restored mechanical and thermal sensation to baseline levels in TG+ mice with SCI. This behavioral profile and responsiveness to chronic training will be important to consider when choosing models to study the mechanisms underlying sensorimotor recovery after SCI.
The C57BL/6 mouse strain is represented by distinct substrains, increasingly recognized to differ genetically and phenotypically. The current study compared stroke vulnerability among C57BL/6 J (J), C57BL/6JEiJ (JEiJ), C57BL/6ByJ (ByJ), C57BL/6NCrl (NCrl), C57BL/6NJ (NJ) and C57BL/6NTac (NTac) substrains, using a model of permanent distal middle cerebral artery and common carotid artery occlusion. Mean infarct volume was nearly two-fold smaller in J, JEiJ and ByJ substrains relative to NCrl, NJ and NTac (N-lineage) mice. This identifies a previously unrecognized confound in stroke studies involving genetically modified strain comparisons if control substrain background were not rigorously matched. Mean infarct size was smaller in females of J and ByJ substrains than in the corresponding males, but there was no sex difference for NCrl and NJ mice. A higher proportion of small infarcts in J and ByJ substrains was largely responsible for both substrain- and sex-dependent differences. These could not be straightforwardly explained by variations in posterior communicating artery patency, MCA anatomy or acute penumbral blood flow deficits. Their larger and more homogeneously distributed infarcts, together with their established use as the common background for many genetically modified strains, may make N-lineage C57BL/6 substrains the preferred choice for future studies in experimental stroke.
Background:
Chronic cerebral hypoperfusion (CCH) is associated with neurological changes and cognitive decline. It is a major cause of vascular dementia and a contributing factor in Alzheimer disease. Animal models are useful in helping to elucidate the mechanisms of these diseases while demonstrating differences in pathological onset and severity. Furthermore, different mouse strains show differences in their susceptibility to neurological damage resulting in different cognitive outcomes.
Purpose:
This study investigated the effect of CCH induced by permanent unilateral common carotid artery occlusion (UCO) on neurological damage in vulnerable brain regions such as hippocampus, striatum, and white matter areas from 2 to 8 weeks following CCH induction.
Methods:
Thirty-six male Institute of Cancer Research (ICR) mice were randomly divided into 2 main experimental groups, Sham and UCO. These 2 main groups were further divided into 3 observation periods of 2, 4, and 8 weeks following CCH. Histological study was then employed using 0.1% cresyl violet and luxol fast blue staining to assess neurological damage.
Results:
We found equal levels of neurological damage induced by CCH between ipsi- and contralateral hemispheres. Hippocampus and striatum damage were slightly increased from 2 to 8 weeks rising to significance at 8 weeks in both areas, while the white matter densities of the corpus callosum, internal capsule, optic tract and striatum fiber did not change.
Conclusion:
CCH induced by UCO in ICR mice induces hippocampal and striatal damage at 8 weeks while leaving white matter undamaged.
Lipocalin-2 (LCN2) has diverse functions in multiple pathophysiological conditions; however, its pathogenic role in vascular dementia (VaD) is unknown. Here, we investigated the role of LCN2 in VaD using rodent models of global cerebral ischemia and hypoperfusion with cognitive impairment and neuroinflammation. Mice subjected to transient bilateral common carotid artery occlusion (tBCCAo) for 50 min showed neuronal death and gliosis in the hippocampus at 7 days post-tBCCAo. LCN2 expression was observed predominantly in the hippocampal astrocytes, whereas its receptor was mainly detected in neurons, microglia, and astrocytes. Furthermore, Lcn2-deficient mice, compared with wild-type animals, showed significantly weaker CA1 neuronal loss, cognitive decline, white matter damage, blood-brain barrier permeability, glial activation, and proinflammatory cytokine production in the hippocampus after tBCCAo. Lcn2 deficiency also attenuated hippocampal neuronal death and cognitive decline at 30 days after unilateral common carotid artery occlusion (UCCAo). Furthermore, intracerebroventricular (i.c.v) injection of recombinant LCN2 protein elicited CA1-neuronal death and a cognitive deficit. Our studies using cultured glia and hippocampal neurons supported the decisive role of LCN2 in hippocampal neurotoxicity and microglial activation, and the role of the HIF-1α-LCN2-VEGFA axis of astrocytes in vascular injury. Additionally, plasma levels of LCN2 were significantly higher in patients with VaD than in the healthy control subjects. These results indicate that hippocampal damage and cognitive impairment are mediated by LCN2 secreted from reactive astrocytes in VaD.
Naturally occurring cell death (NOCD) is a prominent feature of the developing nervous system. During this process, neurons express bcl-2, a major regulator of cell death whose expression may determine whether a neuron dies or survives. To gain insight into the possible role of bcl-2 during NOCD in vivo, we generated lines of transgenic mice in which neurons overexpress the human BCL-2 protein under the control of the neuron-specific enolase (NSE) or phosphoglycerate kinase (PGK) promoters. BCL-2 overexpression reduced neuronal loss during the NOCD period, which led to hypertrophy of the nervous system. For instance, the facial nucleus and the ganglion cell layer of the retina had, respectively, 40% and 50% more neurons than normal. Consistent with this finding, more axons than normal were found in the facial and optic nerves. We also tested whether neurons overexpressing BCL-2 were more resistant to permanent ischemia induced by middle cerebral artery occlusion; in transgenic mice, the volume of the brain infarction was reduced by 50% as compared with wild-type mice. These animals represent an invaluable tool for studying the effects of increased neuronal numbers on brain function as well as the mechanisms that control the survival of neurons during development and adulthood.
Light microscopic neuronal changes were studied in rats subjected to 10 min of global ischemia produced by compression of the major cardiac vessels. Observations of cresyl violet-stained sections revealed early changes involving predominantly GABAergic neurons in various locations. In rats killed 15 min after recirculation, the changes were characterized by the appearance of a clear peripheral zone with condensation of the remaining neuronal cytoplasm. After 1 h, these zones appeared to be compartmentalized into individual pearl-like vacuoles, especially prominent in the nucleus reticularis thalami. After 3 h, the cytoplasmic vacuoles disappeared and the neuronal changes, particularly in the cerebral cortex, striatum, hippocampus, and pars reticulata of the substantia nigra, consisted mainly of hyperchromasia or loss of Nissl substance. After 2 days, the cerebral cortex and thalamus contained occasional neurons with conspicuously large nucleoli. After 7 days, the hippocampus revealed an approximately 50% loss of CA1 pyramidal neurons, associated with intense microglial reactivity in the stratum radiatum, whereas the neuronal destruction was more complete in the nucleus reticularis thalami. Our observations suggest a possibility that early changes in GABAergic neurons may provide a period of neuronal disinhibition and thus contribute to an excitatory ischemic damage in regions connected by GABAergic circuitry.
Free radical-mediated oxidative damage has been implicated in tissue injury resulting from ischemia/reperfusion events. Global cortical ischemia/reperfusion injury to Mongolian gerbil brains was produced by transient occlusion of both common carotid arteries. Protein oxidation, as measured by protein carbonyl content, increased significantly during the reperfusion phase that followed 10 min of ischemia. The activity of glutamine synthetase, an enzyme known to be inactivated by metal-catalyzed oxidation reactions, decreased to 65% of control levels after 2 hr of reperfusion that followed 10 min of ischemia. We also report that the free radical spin trap N-tert-butyl-alpha-phenylnitrone [300 mg/kg (body weight)] administered 60 min before ischemia/reperfusion is initiated, partially prevents protein oxidation and protects from loss of glutamine synthetase activity. In addition, we report a N-tert-butyl-alpha-phenylnitrone-dependent nitroxide radical obtained in the lipid fraction of the ischemia/reperfusion-lesioned brains, but there was very little radical present in the comparable sham-operated control brains. These data strengthen the previous observation utilizing in vivo-trapping methods, that free radical flux is increased during the reperfusion phase of the ischemia-lesioned gerbil brain. The loss of glutamine synthetase would be expected to increase the levels of brain L-glutamate. Thus, the oxidative inactivation of glutamine synthetase may be a critical factor in the neurotoxicity produced after cerebral ischemia/reperfusion injury.
Several laboratories have reported a significant reduction of ischemia-induced injury to hippocampal neurons in rodents treated with competitive and noncompetitive N-methyl-D-aspartate (NMDA) receptor-channel antagonists. This study examined the effects of the noncompetitive antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) in Mongolian gerbils subjected to 5 min of bilateral carotid artery occlusion. In adult female gerbils, single doses of MK-801 injected 1 hr prior to ischemia significantly (p less than 0.01) reduced damage to CA1 hippocampal neurons. However, the drug rendered the postischemic animals comatose and hypothermic for several hours compared with the saline-treated animals. In subsequent experiments, animals pretreated with MK-801 and maintained normothermic during and after forebrain ischemia demonstrated no amelioration of hippocampal damage. Gerbils not treated with MK-801, but kept hypothermic in the postischemic period to approximately the same degree (34.5 degrees C) and duration (8 hr) as was induced by MK-801 therapy showed significant (p less than 0.01) protection of CA1 neurons against ischemia. The neuroprotective activity of MK-801 against transient global ischemia appears to be largely a consequence of postischemic hypothermia rather than a direct action on NMDA receptor-channels.
We have tested whether small intraischemic variations in brain temperature influence the outcome of transient ischemia. To measure brain temperature, a thermocouple probe was placed stereotaxically into the left dorsolateral striatum of rats prior to 20 min of four-vessel occlusion. Rectal temperature was maintained at 36-37 degrees C by a heating lamp, and striatal temperature prior to ischemia was 36 degrees C in all animals. Six animal subgroups were investigated, including rats whose intraischemic striatal brain temperature was not regulated, or was maintained at 33, 34, 36, or 39 degrees C. Postischemic brain temperature was regulated at 36 degrees C, except for one group in which brain temperature was lowered from 36 degrees C to 33 degrees C during the first hour of recirculation. Energy metabolites were measured at the end of the ischemic insult, and histopathological evaluation was carried out at 3 days after ischemia. Intraischemic variations in brain temperature had no significant influence on energy metabolite levels measured at the conclusion of ischemia: Severe depletion of brain ATP, phosphocreatine, glucose, and glycogen and elevation of lactate were observed to a similar degree in all experimental groups. The histopathological consequences of ischemia, however, were markedly influenced by variations in intraischemic brain temperature. In the hippocampus, CA1 neurons were consistently damaged at 36 degrees C, but not at 34 degrees C. Within the dorsolateral striatum, ischemic cell change was present in 100% of the hemispheres at 36 degrees C, but in only 50% at 34 degrees C. Ischemic neurons within the central zone of striatum were not observed in any rats at 34 degrees C, but in all rats at 36 degrees C. In rats whose striatal temperature was not controlled, brain temperature fell from 36 to 30-31 degrees C during the ischemic insult. In this group, no ischemic cell change was seen within striatal areas and was only inconsistently documented within the CA1 hippocampal region. These results demonstrate that (a) rectal temperature unreliably reflects brain temperature during ischemia; (b) despite severe depletion of brain energy metabolites during ischemia at all temperatures, small increments of intraischemic brain temperature markedly accentuate histopathological changes following 3-day survival; and (c) brain temperature must be controlled above 33 degrees C in order to ensure a consistent histopathological outcome. Lowering of the brain temperature by only a few degrees during ischemia confers a marked protective effect.
The present study was undertaken to correlate calcium accumulation with the development of neuronal necrosis following transient ischemia. After 10 min of forebrain ischemia in the rat--a period that leads to reproducible damage of CA1 pyramidal cells--determination of calcium concentration and evaluation of morphological signs of cell body necrosis in the dorsal hippocampus were performed at various recirculation times. Tissue calcium concentration was not different from control at the end of ischemic period and did not change after 3, 6, 12, or 24 h of recirculation. However, after 48 h, calcium content increased significantly, with a further increase being seen after 72 h. At early recovery periods, only scattered necrotic neurons were observed. After 48 h, only 2 of 12 hemispheres showed more than 25 necrotic cells per section. More conspicuous neuronal death was observed after 72 h. The results thus demonstrate that net accumulation of calcium in regio superior of the hippocampus precedes marked necrosis of CA1 pyramidal cells. The results suggest that one primary event in the delayed death of these cells is membrane dysfunction with increased calcium cycling.
The consequences of cerebral ischemia were studied in three different strains (BDF, CFW, and BALE/ C) of mice. The different strains exhibited significant differences in susceptibility to 24-h focal ischemia. Following middle cerebral artery occlusion (MCAO), infarct volumes (mm3) were 5 ± 3 in BDF, 15 ± 5 in CFW, and 23 ± 3 in BALB/C mice (p < 0.05). MCAO plus ipsilateral common carotid artery occlusion (CCAO) resulted in infarct volumes of 15 ±9 in BDF, 38 ±10 in CFW, and 72 ± 12 in BALB/C mice (p < 0.05). In addition, MCAO plus CCAO produced death by 24 h in 42% of CFW and 67% of BALB/C mice, but not in any BDF mice (p < 0.05). CCAO alone produced multifocal hemispheric infarctions in 36% of BALB/C mice but not in the other two strains. Brains of all mouse strains subjected to sham surgery were free of any ischemie injury. Arterial blood pressures, blood gases, and blood cell profiles were relatively similar for the three mouse strains. However, carbon black studies of the cerebrovascular anatomy revealed an incomplete circle of Willis (i.e., a significant decrease in the frequency of patent posterior communicating arteries) for BALB/C compared with BDF mice (p < 0.05), with CFW mice being intermediary. Based on these anatomical data, BALB/C mice also were evaluated following transient global brain ischemia produced by bilateral CCAO. BALB/C mice exhibited a > 85% reduction in cortical microvascular perfusion and EEG power within 1 min of bilateral CCAO. Also, hippocampal neuronal CA1 damage and mortality over 7 days were related to the duration of global brain ischemia (p < 0.05). These data demonstrate a significant difference between mouse strains in their sensitivity to cerebral ischemia that appears to be related, at least in part, to the functional vascular anatomy at the level of the posterior communicating arteries. In particular, we point out the potential usefulness of BALB/C mice as a sensitive and reproducible model of focal and global ischemia.
The proposal that nitric oxide (NO) or its reactant products mediate toxicity in brain remains controversial in part because of the use of nonselective agents that block NO formation in neuronal, glial, and vascular compartments. In mutant mice deficient in neuronal NO synthase (NOS) activity, infarct volumes decreased significantly 24 and 72 hours after middle cerebral artery occlusion, and the neurological deficits were less than those in normal mice. This result could not be accounted for by differences in blood flow or vascular anatomy. However, infarct size in the mutant became larger after endothelial NOS inhibition by nitro-L-arginine administration. Hence, neuronal NO production appears to exacerbate acute ischemic injury, whereas vascular NO protects after middle cerebral artery occlusion. The data emphasize the importance of developing selective inhibitors of the neuronal isoform.
ABSTRACT— A model is described in which transient ischemia is induced in rats anaesthetized with N2O:O2 (70:30) by bilateral carotid artery clamping combined with a lowering of mean arterial blood pressure to 50 mm Hg, the latter being achieved by bleeding, or by bleeding supplemented with administration of trimetaphan or phentolamine. By the use of intubation, muscle paralysis with suxamethonium chloride, and insertion of tail arterial and venous catheters, it was possible to induce reversible ischemia for long-term recovery studies.Autoradiographic measurements of local CBF showed that the procedure reduced CBF in neocortical areas, hippocampus, and caudoputamen to near-zero values, flow rates in a number of subcortical areas being variable. Administration of trimethaphane or phentolamine did not affect ischemic and postischemic flow rates, nor did they alter recovery of EEG and sensory-evoked responses, but trimetaphan blunted the changes in plasma concentrations of adrenaline and noradrenaline.Recovery experiments showed that 10 min of ischemia gave rise to clear signs of permanent brain damage, with a small number of animals developing postischemic seizures that led to the death of the animals in status epilepticus. After 15 min of ischemia, such alterations were more pronounced, and the majority of animals died. It is concluded that the short revival times noted are explained by the fact that the model induces near-complete ischemia, and that recovery following forebrain ischemia is critically dependent on residual flow rates during the period of ischemia.
A new model of transient, bilateral hemispheric ischemia in the unanesthetized rat is described. During ether anesthesia the rat's vertebral arteries were electrocauterized through the alar foramina of the first cervical vertebra and reversible clasps placed loosely around the common carotid arteries. Twenty-four hr later, the awake rats were restrained and the carotid clasps tightened to produce 4-vessel occlusion. The carotid clasps were removed after 10, 20 or 30 min of 4-vessel occlusion and the animals killed by perfusion fixation 72 hr later. Rats which convulsed during the ischemic or post-ischemic period were excluded from further study. All rats subjected to 20 or 30 min of 4-vessel occlusion demonstrated ischemic neuronal damage. The H1 and paramedian hippocampus, striatum and layers 3, 5 and 6 of the posterior neocortex were the regions most frequently damaged. The advantages of this model are the ease of preparation of large numbers of animals, a high rate of predictable ischemic neuronal damage, a low incidence of seizures and the absence of anesthesia.
The duration of survival during a hypoxic or ischemic incident can be altered by barbiturate anesthesia. If this effect on the brain results from a reduction in lactic acid production by hypoxia, then a similar protective effect may be produced by altering substrate availability. Six groups of mice were subjected to hypoxia (4 to 5% O2, balance N2) at 30 to 35°C: 1. Hypoglycemia was induced by 2 U insulin injected ip 30 min prior to hypoxia. 2. Ketotic hypoglycemia was induced by fasting 85 to 90 hours prior to hypoxia. 3. Hyperglycemia was induced by iv dextrose. 4. Diabetic-ketotic-hyperglycemia was induced by iv alloxan 5 days prior to hypoxia. 5. One group was given both the insulin and dextrose in the above sequence. 6. In controls, saline was given iv or ip when appropriate. The mean survival time for ketotic-hypoglycemic and diabetic-ketotic-hyperglycemic mice was significantly higher than control. The mean survival time for the insulin-hypoglycemic mice was significantly lower than control. The remaining groups showed no difference from control. The observed improvement in survival time from hypoxia seen in the ketotic animals suggests that during hypoxia, the brain metabolizes ketones selectively and minimizes the production of lactic acid to maintain neuronal viability.
Cerebral ischemia was produced in gerbils by ligation of the right common carotid artery and the resulting clinical manifestations and pathological alterations, along with electroencephalographic findings, were followed from 30 minutes to 24 hours. Protein synthesis was evaluated with brain slices in vitro and subsequent cellular and subcellular fractionations. One group of animals developed clinical signs of cerebral ischemia and stroke very rapidly and often died within 12 hours. In these animals cerebral infarction was diffuse in the right side of brain within a few hours post-operatively and there was persistent suppression in the electroencephalographic recordings. Amino acid incorporation into protein of subcellular fractions was decreased to 50% of the opposite side at 30 minutes and further declined to less than 10% in 8 to 10 hours. Another group of animals survived to 24 hours in spite of severe neurological manifestations, and protein synthesis was about 15% of the control side at 24 hours. The suppression of protein synthesis was observed both in the neuronal and neurologlial fractions indicating similar vulnerability of these cellular elements toward cerebral ischemia as shown with cerebral anoxia in the past. It was emphasized that the correlation of clinical manifestations and biochemical data is very important to extract meaningful information from biochemical investigations in this model.
The effect of racemic mephobarbital and its optical isomers on survival time of mice exposed to 5% O2 was studied. There was an increase in survival time from 4.2 minutes to 12.6 minutes for 100 mg/kg of the anesthetically active (-) isomer and the racemic form, but no increase for 100 mg/kg of the inactive (+) isomer. Since it has been shown that there is no difference in brain concentrations between the isomers, we conclude that the barbiturate protective effect is bound to the anesthetic effect. All mice convulsed, and since the non-anesthetized animals convulsed earlier and stronger than the anesthetized, it was possible that barbiturate protection was accounted for by its anticonvulsant effects. Diazepam 7.5 mg/kg, while reducing convulsions to the same degree as barbiturates without producing anesthesia, only increased survival time to 6.2 minutes. Thus, the barbiturate protective effect is distinct from the anticonvulsant effect. It seems to be bound to a stereospecific receptor for both protection and anesthesia.
The known susceptibility of the Mongolian gerbil to cerebral infarction following unilateral carotid artery ligation has been attributed in the past to the demonstrated absences of an anastomotic supply between the anterior and posterior cerebral circulations. In a study of 34 adult gerbils exposed to such a procedure, 11, or 33%, developed severe neurological sequelae and succumbed to the procedure in less than 30 hr, whereas 23 animals survived with only minor or transient neurological signs. All animals displayed the expected lack of an anastomosis between the anterior and posterior circulations, but in addition the animals which survived the procedure were found to have a prominent early cross-connection between the anterior cerebral arteries, whereas the animals which succumbed had no such connection. Neuropathological changes in susceptible animals were apparent as early as 3 and one-half hr after ligation and consisted of edema, initially perivascular and then intraneuronal, slowed by acute necrosis. A variety of other vascular anomalies was encountered. We conclude that the peculiar susceptibility of Mongolian gerbils to cerebral infarction following acute unilateral common carotid artery ligation is not related primarily to lack o adequate collaterals between the anterior and the anterior cerebral arteries, but to the degree of adequate adequacy of communication between the anterior cerebral arteries. The critical difference may be more one of degree, i.e. the point at which the medial branches of the anterior cerebral artery fust to become anazygos vessel, rather than an actual difference in the pattern of distribution of the anterior cerebral arteries. The presence of other variation in vascular supply in a relatively small series suggests that results of similar studies of infarction and response to treatment be interpreted with caution.
Oxygen-derived free radicals have been implicated in the pathogenesis of vasogenic edema and infarction caused by ischemia and reperfusion injury. In earlier studies, exogenously supplied liposome-entrapped CuZn superoxide dismutase (CuZn-SOD) ameliorated ischemic brain edema and infarction in rats following focal cerebral ischemia. To ascertain directly the role of SOD in the protection against superoxide radical-induced injury, we measured infarct size and water content 24 hr following focal cerebral ischemia in nontransgenic mice and in transgenic mice bearing the human SOD1 gene. These transgenic mice have 3.1-fold higher cellular CuZn-SOD activity in the brain than do their nontransgenic littermates. We also measured antioxidant levels (reduced glutathione and reduced ascorbate) of contralateral cortex, infarct cortex, surrounding cortex, and striatum. Infarct size and brain edema were significantly decreased in transgenic mice compared with nontransgenic mice. Reduced glutathione and reduced ascorbate levels decreased in the ischemic hemisphere, but levels in surrounding cortex and striatum were significantly higher in transgenic mice than in nontransgenic mice. These results indicate that increased endogenous SOD activity in brain reduces the level of ischemic damage and support the concept that superoxide radicals play an important role in the pathogenesis of infarction and edema following focal cerebral ischemia.
Bilateral occlusion of the carotid arteries (BCAO) killed 52% of the male ddY mice (N = 86) and 77% of the ICR mice (N = 96) within 10 min, and the mean survival time of the ddY strain recorded for the 10 min was significantly longer than the time of the ICR strain. Among animals that survived longer than 1 hr after BCAO, some (5 of ddY and 3 of ICR) were able to survive for more than 24 hr. All of the neurobehavioral and histopathological signs developed by BCAO and in most cases followed by death were found to be also inducible by unilateral occlusion alone, although this was in a small fraction of mice. The brain levels of ATP, glucose and acetylcholine significantly decreased in mice that died within 10 min after BCAO, while none of these changes were detectable in mice surviving BCAO for 1 hr, just as in mice that died by carbon monoxide or ether inhalation. The results obtained herein indicate that mice may not be homogeneous in the functional level of the collateral route of blood supply to the brain tissue and/or in the sensitivity toward the ischemia-inducible lethality.
During anesthesia in mice, both common carotid arteries were tied loosely with an overhand knot suture (an occluder), while two snares (releasers) were placed in the knot so that it could be repeatedly tightened to occlude the arteries and loosened again to allow for reperfusion while the mice were conscious and unrestrained. The incidence of mortality as well as impairment of brain metabolism depended upon the length of cerebral ischemia. Cortical electroencephalogram (EEG) clearly reflected the regional ischemia as evidenced by electrical quiescence. Less mortality was observed with ischemic mice treated with dextrorphan (30 mg/kg p.o.). On day 1 (24 hr after ischemia), there were impairments in complex motor coordination, multichoice swim performance, and step-through or thermal pain-motivated avoidance responses. Thereafter, the battery of tests progressively improved. This improvement depended on the period of resumption of cerebral blood flow; the 7-day, postischemic lapse significantly reduced the deficit observed. Reduction in the degree of habituation of exploratory activity was also clearly observed following ischemic insult. Dextrorphan (1-30 mg/kg i.p.) given to ischemic mice was effective in the habituation and step-through-type passive avoidance test paradigms. In conclusion, 1) the decline in cognition as observed with ischemic mice is due to the temporal and reversible derangement of their neuronal networks; 2) excessively released glutamate is probably of major pathogenic importance in the consequences of cerebral ischemia based on the positive results of the N-methyl-D-aspartate receptor antagonist, dextrorphan; 3) the simple technique could be useful in elucidating the pathophysiologic mechanisms of ischemically elicited derangement of the cerebral organization; and 4) the model could be used to assess the efficiency of drugs with high clinical predictivity.
Evolution, progression and recovery of neural damage during and following cerebral ischemia were investigated in the gerbil after occlusion of a posterior communicating artery and by using the immunohistochemical reaction for tubulin and creatine kinase BB-isoenzyme which are enriched in the neuronal structure and the reaction for astroprotein which is specific for astrocytes. The transcardiac perfusion study with India ink revealed marked hypoperfusion diffusely in the hippocampus and moderately in the thalamus on the occluded side. The earliest immunohistochemical lesion, manifested as loss of the reaction for tubulin and creatine kinase BB-isoenzyme in dendrites and nerve cell bodies, was found in the CA1 and CA2 region of the hippocampus after ischemia for 4 min, while it took 10 min before the earliest lesion became visible in the ventral nucleus of the thalamus and it took over 1 h before scattered lesions evolved in granular cells of the dentate gyrus. The staining with hematoxylin-eosin was much less sensitive in detection of early ischemic lesions. After re-establishment of blood flow to the posterior communicating artery, the ischemic lesions which were visualized with the reaction for tubulin or creatine kinase BB-isoenzyme disappeared or reduced the size, if the ischemic period was brief. Beyond a certain ischemic period, the lesion expanded further during the early postischemic period. The reaction for astroprotein visualized reactive astrocytes even in the area without any abnormalities with other reactions, an evidence of subtle ischemic insults.
Experimental cerebral ischemia was produced in gerbils by occlusion of the right common carotid artery in the neck. The evolution of the ischemic lesions was followed from five minutes to six hours by using the immunohistochemical techniques for tubulin and creatine kinase BB-isoenzyme. The earliest lesion was found in the subiculum-CA1 and CA2 regions of the hippocampus in five minutes. There was loss of staining in the apical dendrites and perikarya of the pyramidal cells. The earliest lesion in the cerebral cortex, visible in ten minutes, was a laminar loss of staining for tubulin. Evolution of the ischemic lesions in the thalamus and caudoputamen was delayed. However, in two hours widespread ischemic lesions were seen there. Evolution of the ischemic lesions was slightly slower with the reaction for creatine kinase BB-isoenzyme as compared to the reaction for tubulin, but was far more sensitive than hematoxylin-eosin staining. The distribution of ischemic lesions detected by the immunohistochemical method compared to ischemic areas detected by an India ink perfusion study suggested that both the extent of regional ischemia and regional difference in tissue vulnerability were contributing factors for the emergence of early ischemic lesions. The mechanism for prompt disappearance of the immunohistochemical reaction for tubulin is not clear, but the present investigation demonstrates the usefulness of the immunohistochemical technique for detecting early ischemic lesions and provides a possible biochemical mechanism for cellular damage after ischemic insults.
A simple and reliable method for the preparation of an ischemia-induced amnesic model was developed in mice and the model animals were employed for screening of the anti-amnesic effect of drugs. Under light thiopental anesthesia the bilateral carotid arteries were exfoliated from the surrounding tissues. Each artery was threaded through a small polyethylene tube, and the incision was sutured, leaving the tip of the tube and both ends of the thread out of the skin. To prevent the tube from falling off, both ends of the thread was ligated at the tip of the tube. Cerebral ischemia was attained by pulling the artery into the tube, and the occlusion was rapidly released by cutting the ligation in the thread and taking off the tube. Twenty-four hours after the surgery, the animals were used for the one trial passive avoidance learning test. In ddY strain mice, the amnesic state was obtained by 5 to 30 min cerebral ischemia, which was applied immediately after the acquisition trial. Mice of the ICR strain were more sensitive to the ischemic treatment and 2 min occlusion of the carotid arteries resulted in a high degree of amnesia. A potent prolyl endopeptidase inhibitor, Z-thiopro-thiazolidine, and 2 novel pyrrolidone derivatives, p-chlorobenzyl-2-pyrrolidone-5-carboxylate and N-(2-pyridylmethyl)-2-pyrrolidone-5-carboxamide, improved the acquisition of the passive avoidance response in the ischemia-induced amnesic models.
We investigated the neuronal distribution of microtubule-associated protein 2 in gerbil brain and monitored the progression of ischemic damage immunohistochemically by using this protein as a dendritic marker. The reaction for microtubule-associated protein 2 in normal gerbil brain clearly visualized neuronal soma and dendrites but other structures such as axonal bundles, glia and endothelial cells exhibited little immunoreactivity. In a reproducible gerbil model of unilateral cerebral ischemia, we could detect the ischemic lesions as early as 3 min after right common carotid occlusion at the subiculum-CA1 region of the ipsilateral hippocampus as faint loss of the reaction in the dendrites. After ischemia for 30 min, the ischemic lesions were clearly detected as loss of the reaction in the nerve cell bodies, dendrites and the neuropil in the hippocampus, cerebral cortex, thalamus and the caudoputamen. Although the mechanism for prompt disappearance of the immunohistochemical reaction for microtubule-associated protein 2 is not clear, the present investigation suggests that dendrites in the vulnerable regions may be quite susceptible to ischemic stress and that the immunohistochemical procedure for microtubule-associated protein 2 may be very useful for demonstration of dendritic damage in various pathophysiological states of the central nervous system.
The changes in cerebral metabolism in mice in severe hypoxia were investigated by analyses of changes in the levels of energy metabolites and near-infrared spectrophotometric assessment of the states of hemoglobin and cytochrome oxidase. Under 4.4% O2, the contribution of anaerobic ATP production was at most about 20% of the demand. However, the cerebral ATP level was kept at the control level until about 1 min before death. Pentobarbital anesthesia, which reduced the cerebral rate of metabolism, prolonged the survival time, although anaerobic ATP production still did not support ATP demand. Under these conditions, deoxygenation of hemoglobin and reduction of cytochrome oxidase proceeded rapidly within 1 min. Hemoglobin reached the maximum state of deoxygenation in the middle phase of hypoxia, with no further change until death. However, cytochrome oxidase was reduced slowly with one phase of partial reoxidation due to increase of cerebral blood volume, and reached the completely reduced state at death. From these results it was concluded that the aerobic ATP synthesis, which supplied more than 80% of the cerebral demand, decreased gradually because of limitation of oxygen supply, and that the failure of oxidative phosphorylation to meet demand triggered the decrease in the cellular ATP level that led to death.
We investigated progression and recovery of neuronal damage during and after global cerebral ischemia in gerbils after bilateral occlusion of the common carotid arteries, using the immunohistochemical method (reaction for tubulin and creatine kinase BB-isoenzyme). The earliest, but reversible, ischemic lesions occurred after 3 minutes' ischemia in the subiculum-CA1 and CA2 regions of the hippocampus. The lesions became irreversible after 4 minutes' ischemia. The ischemic and postischemic lesions in the cerebral cortex, thalamus, and caudoputamen were partially or completely reversible if the ischemic period was 5 minutes, whereas delayed degeneration occurred in the pyramidal cells of the medial CA1 region after reperfusion for 48 hours (delayed neuronal death). After 10 minutes' ischemia and subsequent reperfusion, delayed neuronal death extended from the medial to the lateral CA1 region; the ischemic and postischemic lesions in the cerebral cortex, thalamus, and caudoputamen also expanded during reperfusion. Our investigation demonstrates that selective vulnerability existed in global cerebral ischemia as in incomplete or regional ischemia and suggests that neurons in many areas of the brain possessed the potential for recovery, progressive deterioration, and even delayed neuronal death depending on the severity and duration of cerebral ischemia.
A method was developed to predict the severity of cerebral ischemia before permanent occlusion of a common carotid artery in gerbils by observing the diameter and appearance of the artery after temporary occlusion and observing clinical signs after permanent occlusion. The severity of cerebral ischemia was confirmed by a sensitive immunohistochemical method and measurement of focal cerebral blood flow after 30 minutes' ischemia. All gerbils with greater than 40% reduction of the diameter and a white arterial margin distal to temporary occlusion developed severe neurologic signs following permanent occlusion, but no gerbils with reduction of less than 30% and a red arterial margin developed neurologic signs. With the cumulative neurologic score, gerbils could be divided into classes with no, mild, moderate, and severe symptoms, mostly after 10 minutes. Severely symptomatic gerbils were identified in 3 minutes. Extensive ischemic damage was observed in severely symptomatic gerbils, but no immunohistochemical lesion was detected in mildly symptomatic gerbils. Cerebral blood flow was markedly reduced in severely symptomatic gerbils but more selectively reduced in the cortical structures of moderately symptomatic gerbils. This prediction method is useful for investigating early cerebral ischemia and for evaluating the effectiveness of pharmacologic agents.
The present study was designed to elucidate whether naftidrofuryl oxalate (LS-121) may exert a beneficial effect on survival time and cerebral energy metabolism of bilateral carotid artery (BCA)-ligated mice. Survival time of BCA-ligated mice ranged from 100 to 308 sec. Administration of 15, 45 and 100 mg/kg, i.p. and 100 mg/kg, p.o. LS-121 significantly prolonged the survival time. Cerebral adenosine triphosphate (ATP), creatine phosphate (CP) and glucose contents were markedly reduced at 2 min after BCA-ligation. Cerebral lactate content was increased by the ligation, whereas the pyruvate content was not altered. Pretreatment of mice with 15, 45 mg/kg, i.p. and 100 mg/kg, p.o. LS-121 suppressed BCA-ligation induced decrease in high-energy phosphates of the mouse brain. Ligation-induced decrease in glucose and increase in lactate content tended to be attenuated by the treatment with 45 mg/kg, i.p. LS-121. Time course of changes in metabolic variables altered by BCA-ligation with and without the pretreatment with 45 mg/kg, i.p. LS-121 showed a significant suppression of ligation-induced decrease in cerebral high-energy phosphates. The results suggest that LS-121 is beneficial for ischemic mouse brain energy metabolism, which may be related to the prolongation of the survival time.
Brain levels of NADH and NAD+ were measured in three models of cerebral ischemia to determine whether degradation of the pyridine nucleotides is enhanced in models that generate high concentrations of lactic acid. Complete ischemia (decapitation), in which lactate increased to 14 mmol/kg, caused a gradual decrease in the NAD pool to 50% of control by 2 h. During focal ischemia (occlusion of the middle cerebral artery), the decrease in the NAD pool was less pronounced (82% of control at 2 h) despite the accentuated accumulation of lactate to 33 mmol/kg. In a third model (unilateral hypoxia-ischemia), pretreatment of animals with glucose augmented the ischemic elevation of lactate from 30 mmol/kg to 40 mmol/kg and greatly impaired restoration of energy metabolites during recirculation. However, glucose pretreatment had no effect on the size of the NAD pool during ischemia or early recovery. These results, therefore, demonstrate that the pyridine nucleotide pool is not rapidly degraded during ischemic insults that accumulate high concentrations of lactic acid. The stability of the NAD pool may have been enhanced by the limited increase in brain levels of NADH that occurred in these models of incomplete ischemia.
Light and electron microscopy has been used to study the cytopathological changes in the rat hippocampus directly after a 30-min period of forebrain ischemia and after 30 or 120 min of reperfusion. The fine structural localization of calcium has been demonstrated using the oxalate/pyroantimonate procedure. Cellular changes considered typical of ischemia (swelling of astrocytic processes, distention of mitochondria, condensation of cytoplasm, "ischemic cell change") are most prominent after 30 min of reperfusion. At this time, dense calcium pyroantimonate deposits are evident in swollen mitochondria in pyramidal and hilar neurons. After 120 min of reperfusion, substantial restitution has occurred; most mitochondria appear normal and there are few calcium deposits. However, a small number of selectively vulnerable neurons (hilar and pyramidal neurons) show dense condensation (ischemic cell change) with multiple vacuoles containing calcium deposits. The role of excessive calcium entry and mitochondrial calcium overload during the reperfusion period in determining the death of selectively vulnerable neurons is discussed.
It has been hypothesized that ischemia, followed by reperfusion, facilitates peroxidative free-radical chain processes in brain. To resolve this question, rats were subjected to reversible global ischemia. From coronal sections of brains frozen in situ, small (ca. 2 mg) amounts of tissue were sampled from neocortex, hippocampus, and thalamus of both cerebral hemispheres of four groups of rats exposed to 30 min cerebral ischemia followed by 0, 30, 60, and 240 min of reperfusion, and from a control group subjected to the same operative procedures, except for the induction of ischemia. Heptane-solubilized total lipid extracts from these samples were analyzed spectroscopically in the 190-330 nm range for content of isolated (nonconjugated) double bonds and of conjugated diene structures; the latter are formed from isolated double bonds during peroxidation of unsaturated fatty acids. Spectra derived from tissue regions of rats subjected to ischemia, or ischemia followed by reperfusion, were compared to averaged, region-specific control spectra and were normalized to the original content of isolated double bonds in the peroxidized samples. The resultant difference spectra were analyzed in terms of ratios of conjugated diene concentration to the concentration of isolated double bonds originally at risk in the specific tissue zones considered. The peak representing conjugated diene formation was centered at 238 +/- 1 nm and was usually well resolved when the molar ratio [conjugated diene]/[isolated double bonds], expressed as a percentage [( CD]/[IDB]), was greater than 0.25%.(ABSTRACT TRUNCATED AT 250 WORDS)
In the CA1 subfield of the gerbil hippocampus, an unusual series of changes were noticed after ischemia. Mongolian gerbils were subjected to bilateral carotid occlusion for 5 min. Perfusion fixation was performed 3, 6 and 12 h or 1, 2, 4, 7 and 21 days afterwards. Specimens obtained from the dorsal hippocampus were processed for light and electron microscopy. Three different types of changes were observed in the CA4, CA2 and CA1 subfields. In CA4, the change was rapid and corresponded to ischemic cell change. The alteration in CA2 was relatively slow, and identical to what has been called reactive change. On the contrary, the change in the CA1 pyramidal cells was very slow, only becoming apparent by light microscopy 2 days following ischemia. The CA1 subfield was selected for electron microscopic observation. The lamellar alignment of proliferated cisterns of the endoplasmic reticulum was the most conspicuous finding in these cells. Four days following ischemia, almost all of the pyramidal cells in CA1 were destroyed. In the CA1 neuropil, numerous presynaptic terminals remained without being apposed to normal postsynaptic sites. These changes in CA1, called here 'delayed neuronal death', may differ from those thought to be typical of ischemic neuronal damage. It was unlikely that the disturbance of local blood vessels was the cause of these changes.
This study examined the temporal profile of ischemic neuronal damage following transient bilateral forebrain ischemia in the rat model of four-vessel occlusion. Wistar rats were subjected to transient but severe forebrain ischemia by permanently occluding the vertebral arteries and 24 hours later temporarily occluding the common carotid arteries for 10, 20, or 30 minutes. Carotid artery blood flow was restored and the rats were killed by perfusion-fixation after 3, 6, 24, and 72 hours. Rats with postischemic convulsions were discarded. Ischemic neuronal damage was graded in accordance with conventional neuropathological criteria. Ten minutes of four-vessel occlusion produced scattered ischemic cell change in the cerebral hemispheres of most rats. The time to onset of visible neuronal damage varied among brain regions and in some regions progressively worsened with time. After 30 minutes of ischemia, small to medium-sized striatal neurons were damaged early while the initiation of visible damage to hippocampal neurons in the h1 zone was delayed for 3 to 6 hours. The number of damaged neurons in neocortex (layer 3, layers 5 and 6, or both) and hippocampus (h1, h3-5, paramedian zone) increased significantly (p less than 0.01) between 24 and 72 hours. The unique delay in onset of ischemic cell change and the protracted increase in its incidence between 24 and 72 hours could reflect either delayed appearance of ischemic change in previously killed neurons or a delayed insult that continued to jeopardize compromised but otherwise viable neurons during the postischemic period.
The proposal that nitric oxide (NO) or its reactant products mediate toxicity in brain remains controversial in part because
of the use of nonselective agents that block NO formation in neuronal, glial, and vascular compartments. In mutant mice deficient
in neuronal NO synthase (NOS) activity, infarct volumes decreased significantly 24 and 72 hours after middle cerebral artery
occlusion, and the neurological deficits were less than those in normal mice. This result could not be accounted for by differences
in blood flow or vascular anatomy. However, infarct size in the mutant became larger after endothelial NOS inhibition by nitro-L-arginine
administration. Hence, neuronal NO production appears to exacerbate acute ischemic injury, whereas vascular NO protects after
middle cerebral artery occlusion. The data emphasize the importance of developing selective inhibitors of the neuronal isoform.
Apoptosis or programmed cell death may be involved in neuronal death in the cerebral cortex after a permanent focal ischemic insult. Studies indicate that protein p53 is a major determinant of the cellular mechanism that leads to programmed cell death. Wild-type C57 mice and two groups of transgenic C57 mice, one homozygous and the other heterozygous for a p53 null gene, were subjected to middle cerebral artery occlusion. As expected, the wild-type mice had a large, consistent infarct volume (22.11 +/- 4.59 mm3; n = 10). Both transgenic groups had significantly less ischemic damage than the wild-type control group. However, unexpectedly, the heterozygous group had the least amount of ischemic damage (16.12 +/- 1.71 mm3, n = 11; 27% reduction in infarct size). The ischemic damage in the homozygous group (18.72 +/- 3.48 mm3, n = 9) was significantly less than in the wild-type control (15% reduction in infarct size) but significantly more than in the heterozygous group. Thus, although the absence of p53 expression was protective, greater protection was afforded by reduced expression of p53. These data suggest that attenuated p53 expression may be protective after an ischemic event.
Naturally occurring cell death (NOCD) is a prominent feature of the developing nervous system. During this process, neurons express bcl-2, a major regulator of cell death whose expression may determine whether a neuron dies or survives. To gain insight into the possible role of bcl-2 during NOCD in vivo, we generated lines of transgenic mice in which neurons overexpress the human BCL-2 protein under the control of the neuron-specific enolase (NSE) or phosphoglycerate kinase (PGK) promoters. BCL-2 overexpression reduced neuronal loss during the NOCD period, which led to hypertrophy of the nervous system. For instance, the facial nucleus and the ganglion cell layer of the retina had, respectively, 40% and 50% more neurons than normal. Consistent with this finding, more axons than normal were found in the facial and optic nerves. We also tested whether neurons overexpressing BCL-2 were more resistant to permanent ischemia induced by middle cerebral artery occlusion; in transgenic mice, the volume of the brain infarction was reduced by 50% as compared with wild-type mice. These animals represent an invaluable tool for studying the effects of increased neuronal numbers on brain function as well as the mechanisms that control the survival of neurons during development and adulthood.
Cerebral intraventricular infusion of acidic or basic fibroblast growth factor has been shown to attenuate ischemic damage to hippocampal CA1 neurons in the gerbil. The purpose of the present study was to determine if the basic fibroblast growth factor transgenic mouse has an enhanced ability to resist the effects of severe cerebral hypoxemia-oligemia.
Mice that were transgenic for bovine basic fibroblast growth factor were exposed to right carotid artery ligation, hyperglycemia, and 20 minutes of 1% carbon monoxide. After 5 days' recovery, brains were examined for histological damage.
Counts of CA1 neurons in the right hippocampus showed a significantly higher number of neurons per millimeter CA1 in hypoxic-ischemic transgenic mice compared with nontransgenic controls (transgenic, 260 +/- 33; nontransgenic, 151 +/- 37 neurons per millimeter CA1; P < .05).
The results indicate that basic fibroblast growth factor transgenic mice, as judged by CA1 hippocampal neuronal survival, have an enhanced ability to resist the effects of a complex hypoxic-ischemic cerebral insult.
Calcium/calmodulin-dependent protein kinase II (CaM-kinase) is a central enzyme in regulating neuronal processes. Imbalances in the activity and distribution of this enzyme have been reported following in vivo ischemia, and sustained decreases in activity correlate with subsequent neuronal death. In this report, mice that had been rendered deficient in the alpha subunit of CaM-kinase using gene knock-out technology were utilized to determine whether this enzyme is causally related to ischemic damage. Using a focal model of cerebral ischemia, we showed that homozygous knock-out mice lacking the alpha subunit exhibited an infarct volume almost twice that of wild-type litter mates. Heterozygous mice exhibited slightly less damage following ischemia than did homozygous mice, but infarct volumes remained significantly larger than those of wild-type litter mates. We conclude that reduced amounts of the alpha subunit of CaM-kinase predisposes neurons to increased damage following ischemia and that any perturbation that decreases the amount or activity of the enzyme will produce enhanced susceptibility to neuronal damage.
To address the importance of nitric oxide or its reaction products as mediators of neurotoxicity in brain, tissue injury was assessed after transient global ischemia in mice rendered mutant in the gene for neuronal nitric oxide synthase. Halothane-anesthetized wild type and mutant mice were subjected to temporary occlusion of the basilar plus both carotid arteries for 5 or 10 min followed by three days of reperfusion. Hippocampal injury, assessed both by qualitative grading and by cell counting in the CA1 subregion, was significantly less in the mutant mice group after 5 or 10 min of ischemia. Mutant mice exhibited a lower mortality (P < 0.01), less weight loss, more normal grooming and spontaneous motor activity and better grasping in the 10 min group. There were no obvious differences in cerebrovascular anatomy or hemodynamics between wild type and mutant mice. The data suggest that a deficiency of neuronal nitric oxide synthase confers increased resistance to transient global cerebral ischemia, and support the suggestion that selective neuronal nitric oxide synthase inhibitors might reduce tissue injury associated with global cerebral ischemia.
Ischemic tolerance following transient global cerebral ischemia has drawn considerable attention because of the putative cell defense mechanism which may be inducible by ischemic stress. The purpose of this study is to investigate the inducibility of ischemic tolerance in moderately symptomatic gerbils after unilateral carotid occlusion. Adult Mongolian gerbils were used. Under ether inhalation, the right common carotid artery was occluded for up to 30 min with an aneurysmal clip. Immediately after occlusion, neurological signs and motor function were evaluated and gerbils with moderate signs were selected for investigation of ischemic tolerance. Ischemia for 30 min to gerbils with moderate signs constantly caused neuronal death in the caudoputamen, but it was prevented by pretreatment with 10 min ischemia which was reversible but strong enough to produce heat shock protein 70. The results show that ischemic tolerance can be induced after hemispheric cerebral ischemia as in the case of global cerebral ischemia and suggest that ischemic tolerance may be relevant in human stroke.
A model of ischemia by carotid occlusion in mice: Assessing neurodegenerative and behavioral effects
- Hengemihle
A new model of bilateral hemispheric ischemia in the unanesthetized rat
- Pulsinelli
Cerebral ischemia model with conscious mice
- Himori
A convenient cerebral ischemic model using mice
- Izumi
Models for studying long-term recovery following forebrain ischemia in the rat. 2. A 2-vessel occlusion model
- Smith