Ischemia-induced slowly progressive neuronal damage in the rat brain

doi:10.1016/0306-4522(90)90378-H

Neuroscience

Volume 38, Issue 1, 1990, Pages 115-124

https://doi.org/10.1016/0306-4522(90)90378-H Get rights and content

Abstract

Ischemic neuronal damage has been believed to make rapid progress in the course of a few days even in delayed selective neuronal death, to say nothing of acute brain necrosis. In the present study, however, we demonstrate for the first time a new type of ischemia-induced neuronal damage which progresses in the course of several weeks or a few months and we tentatively call this process “slowly progressive neuronal damage”.

We have focused on the chronological changes of neuronal damage in the dorsolateral striatum and neocortex following various durations of transient middle cerebral artery occlusion, which does not cause cerebral infarction and is clinically designated “transient ischemic attack”. In the rats subjected to 15 min middle cerebral artery occlusion, the neocortex and lateral striatum were rarely damaged, whereas the small to medium-sized neurons only in the narrow area restricted to the dorsal striatum showed slowly progressive neuronal damage. Prolongation of ischemic duration to 30 min accelerated the evolution of neuronal damage in the dorsolateral striatum and also extended the distribution of neuronal damage to the neocortex, especially to layer III and more superficial layers. Further prolongation of ischemie duration to 45 min resulted in more rapid progress of selective neuronal death in those areas described above, whereas no animal escaped 60 min ischemia, without acute total tissue necrosis in the middle cerebral artery territory.

Ischemia-induced slowly progressive neuronal damage may be implicated in the pathogenesis of such slowly progressive neurologic deterioration as dementia or Parkinsonism in patients with cerebral arteriosclerosis.

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Citation Excerpt :
These morphological changes might due to the neuronal cell death after the ischemia/reperfusion injury and ET-1over-expression further exacerbated the damage. In addition, Nakano has described a phenomenon of “slowly progressive neuronal damage” after short-term ischemia (Nakano, Kogure, & Fujikura, 1990). The delayed neuronal damage in the brain was observed weeks or months after 15 to 45 min MCAO (Nakano et al., 1990), in contrast to the previous opinion that this process only continues for several days after transient ischemia injury (Kirino, 1982; Pulsinelli, Brierley, & Plum, 1982).
Increased level of endothelin-1 (ET-1), a potent vasoconstrictor, has been found in the cerebral spinal fluid (CSF) of patients with multi-infarction dementia, suggesting a possible role of ET-1 in cognitive deficit associated with stroke. Previously, we have reported that synthesis of ET-1 is induced in endothelial cells in hypoxic/ischemic conditions. Transgenic mice over-expressing endothelin-1 in endothelial cells (TET-1) developed systemic hypertension and showed more severe brain damage after transient ischemia. To further understand the significance of endothelial ET-1 in cognitive deficit, we subjected adult TET-1 mice to 30 min middle cerebral artery occlusion (MCAO) with 7 days reperfusion. At baseline, TET-1 mice showed similar locomotor activity, emotion and cognitive function compared to non-transgenic (NTg) mice. However, after 30 min MCAO and 7 days reperfusion, although the sensorimotor function measured by neurological scores was recovered in both genotypes, TET-1 mice showed increased anxiety-like behavior in the open field test and impaired spatial learning and reference memory in the Morris water maze. Parallel with these behavioral changes, TET-1 mice showed more severe brain damage with blood–brain–barrier breakdown (BBB), reactive astrogliosis, increased caspase-3, and increased peroxiredoxin 6 (Prx6) expressions around blood vessels in the ipsilateral hippocampus, compared to that of NTg mice, suggesting that ET-1 over-expression in the endothelial cells leads to more severe BBB breakdown and increased oxidative stress which may resulted in neuronal apoptosis and glial reactivity, which might contribute to the emotional changes and cognitive deficits after short-term ischemia with long-term reperfusion.
Characterizing infarction and selective neuronal loss following temporary focal cerebral ischemia in the rat: A multi-modality imaging study
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This diversity of local tissue outcome likely reflects inter- and intra-subject differences in severity of hypoperfusion during MCAo, previously documented in this model (Buchan et al., 1992; Kaplan et al., 1991) including by us (Hughes et al., 2010). Following proximal tMCAo, SNL is commonly observed in the striatum (Fujioka et al., 1999; Garcia et al., 1997; Katchanov et al., 2003; Li et al., 1999, 2000; Sicard et al., 2006a; Wegener et al., 2006; Winter et al., 2005), but is milder and much less consistent in the neocortex (Garcia et al., 1997; Lehrmann et al., 1997; Li et al., 1999; Nakano et al., 1990; Sicard et al., 2006a), where it tends to affect the borders of the infarct (Garcia et al., 1997; Mies et al., 1983; Nedergaard, 1987). In non-human primates (DeGirolami et al., 1984) and man (Nedergaard et al., 1986; Torvik and Svindland, 1986), definite SNL has so far been observed only as an inconsistent rim bordering the infarct, though this may reflect among other factors biases in duration of ischemia and occurrence of reperfusion, as well as lack of IHC analysis.
Current models dictate that, depending on occurrence of early reperfusion, the ischemic penumbra either undergoes or escapes infarction (i.e., “pan-necrosis”). However, tissue outcome following temporary middle-cerebral artery occlusion (tMCAo) in rodents can also include selective neuronal loss (SNL), which even if subtle may impede functional recovery. In order to explore the pathophysiology of ischemic stroke, determine potential therapeutic targets and monitor effects of therapy, in vivo imaging surrogates of these varied histopathological outcomes applicable in the clinical setting would be useful. Although hyperintense signal on T₂-weighted MRI in the chronic post-stroke stage is considered a reliable surrogate of tissue infarction, SNL is not associated with T₂W abnormal signal. In the clinical setting, the neuron-specific PET ligand ¹¹C-flumazenil (FMZ) has been used to identify both pan-necrosis and peri-infarct SNL, but this inference has not been histopathological confirmed so far. Here we investigated the late tissue sequelae of tMCAo in the rodent using in vivo T₂W MRI and FMZ-PET against post mortem immunohistochemistry as gold standard.
Adult spontaneously hypertensive rats (SHRs) underwent 45 min distal-clip middle-cerebral artery occlusion and, 28 days later, FMZ-PET and T₂W-MRI, immediately followed by immunohistochemistry for neuronal loss (NeuN), activated microglia and astrocytosis. Based on standard histopathological definitions, ischemic lesions were classified into pan-necrosis, partial infarction or SNL. NeuN changes and FMZ binding across the whole hemisphere were quantified in the same set of 44 regions-of-interest according to previously validated protocols; linear regressions between these two measures were carried out both within and across subjects.
Both cortical pan-necrosis/partial infarction and SNL were present in all rats except one, where SNL was isolated and extensive. Infarction/partial infarction, but not SNL, was associated with T₂W hyperintense signals and cortical atrophy. In contrast, FMZ binding was decreased in all types of lesions including SNL, in proportion with NeuN staining intensity both within (p < 0.05 to < 0.001) and across (p < 0.001) subjects, including the subject that showed pure SNL (p = 0.01).
This novel study revealed three main facts: i) long-term histopathological cortical changes following 45 min tMCAo in SHRs included all three of SNL, partial infarction and frank infarction; ii) T2W MRI showed conspicuous high signal lesions for complete or partial infarction, but no changes for SNL; and iii) FMZ-PET was sensitive to all three types of tMCAo-induced histopathological changes, including isolated SNL, suggesting it is a valid surrogate for the histological sequelae of focal cerebral ischemia. In addition, the finding of almost universal completed cortical infarction at 28 days differed from our previous findings at 14-day survival using the same model and rat strain, where SNL was the almost exclusive outcome, possibly representing delayed infarct maturation. Prospective studies are needed to investigate this interesting possibility.
Therapeutic neuroprotective effects of ginkgolide B on cortex and basal ganglia in a rat model of transient focal ischemia
2011, European Journal of Pharmaceutical Sciences
Citation Excerpt :
Later than 2 h after MCAO, has only the chance to protect cortical tissue, which is viable for up to 12–24 h after focal ischemia (Garcia, 1992). Nakano et al. (1990) studied neuronal damage in the dorsolateral striatum and neocortex following various ischemia time subjected in rats, and some articles reported slowly progressive neuronal damage after MCAO. It remains unclear how the ischemic injury progress in different brain regions after MCAO and whether ischemic injury could be reversible after ischemia injury.
Cerebral ischemia and reperfusion is one of the leading causes for death and severe disabilities in the world and often lead to irreversible brain damage over later lifespan. The aim of this study was to investigate the evolution of pathological damage in cerebral cortex and basal ganglia following ischemia and to evaluate the therapeutic neuroprotective effect of ginkgolide B in a rat model of stroke induced by middle cerebral artery occlusion (MCAO). TTC stain, brain water content and Evans-Blue extravasation were used to quantify brain damage. Our results demonstrated that basal ganglia undergo progressive pathological damage earlier following MCAO, and injury was stable and irreversible after 5 h following ischemia. However, onset of ischemia injury in cerebral cortex appeared later than basal ganglia and became evident about 3 h following MCAO, and injury was stable and irreversible after 6 h following ischemia. Blood brain barrier opened progressively, and it seemed to be significantly destroyed after 4 h following MCAO comparing with 0 h. Post-ischemic treatment with ginkgolide B improved neurological function and reduced infarct size in basal ganglia within 3 h and cerebral cortex within 5 h following MCAO. The therapeutic effect of ginkgolide B on extenuate brain edema and decrease blood brain barrier permeability were extended for 5 h after ischemia, and more evident reversal effect were observed when administrated at earlier time.
Propofol improved neurobehavioral outcome of cerebral ischemia-reperfusion rats by regulating Bcl-2 and Bax expression
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Propofol is an intravenous anesthetic with neuroprotective effects against cerebral ischemia–reperfusion (I/R) injury. Few studies regarding the neuroprotective and neurobehavioral effects of propofol have been conducted, and the underlying mechanisms are still unclear. Because I/R may result in neuronal apoptosis, the apoptosis regulatory genes B-cell leukemia-2 (Bcl-2) and Bcl-2-associated X protein (Bax) may be involved in the neuroprotective process. In this study, 120 Wistar rats were randomly divided into three groups (sham, I/R-induced, and propofol-treated). Cerebral ischemia was induced by clamping the bilateral common carotid arteries for 10 min. Propofol (1.0 mg/kg/min) was administered intravenously for 1 h before the induction of ischemia. Neuronal damage was evaluated by neurobehavioral scores and histological examination of the brain sections at the level of the dorsal hippocampus at 6 h, 24 h, 48 h, 72 h, 4 days, 5 days, 6 days, and 7 days after I/R. The apoptotic rate of hippocampal neurons was detected by flow cytometry. The expression of Bcl-2 and Bax was evaluated using immunohistochemical and Western blot methods. The results of this study showed that neurobehavioral scores were higher in propofol-treated rats compared with I/R-induced rats with no propofol treatment. Moreover, the hippocampal expression of Bcl-2 was significantly higher, while the expression of Bax was significantly lower in propofol-treated rats compared with I/R-induced rats at 24 h after ischemia. Hence, this study suggests that the neuroprotective effects of propofol against neuronal apoptosis may be a consequence of the regulation of Bcl-2 and Bax.
Mapping selective neuronal loss and microglial activation in the salvaged neocortical penumbra in the rat
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Citation Excerpt :
With brief proximal tMCAo, SNL has been frequently reported in the striatum (Fujioka et al., 1999; Garcia et al., 1997; Katchanov et al., 2003; Li et al., 1999, 2000; Sicard et al., 2006; Wegener et al., 2006). Mild degrees of neocortical SNL have also been occasionally reported after brief proximal tMCAo (Garcia et al., 1997; Lehrmann et al., 1997; Li et al., 1999; Nakano et al., 1990; Sicard et al., 2006). For instance, cortical “selective neuronal necrosis” was found by Garcia et al. (1997) to affect 40% of rats subjected to 10–25 min thread tMCAo; in this study, dead neurons were only seen using H&E within 7 days of the insult, while after this date only vanishing neuron “ghosts” were barely identifiable.
Rescuing the ischemic penumbra from infarction is the mainstay of acute stroke therapy. However, the rescued penumbra may be affected by selective neuronal loss (SNL) and microglial activation (MA), which may hinder functional recovery and hence represent potential new therapeutic targets. Imaging them in vivo is currently attracting considerable interest, but relevant rat models are needed to underpin methods development and validation. Although striatal SNL/MA is well described following proximal MCA occlusion (MCAo), neocortical SNL/MA is still poorly characterized, yet has greater clinical relevance. This study aimed to assess the distribution and intensity of neocortical SNL and MA in a distal clip MCAo model known to cause severe neocortical ischemia. Spontaneously hypertensive rats were subjected to 45 min distal MCAo with ipsilateral common carotid artery occlusion. At day 14, post mortem SNL and MA were mapped using NeuN and OX42 immunohistochemistry, respectively. In a separate group, cerebral blood flow (CBF) was mapped during MCAo using ¹⁴C-iodoantipyrine autoradiography. Values for SNL, MA, and CBF were obtained in the same set of anatomical ROIs covering the cortical MCA territory. Extensive SNL and MA affected the non-infarcted MCA cortex, adopting a well-defined regional distribution and a striking patchy/pseudo-columnar pattern. Regional intensities of SNL and MA were strongly inter-correlated, and also strikingly related to occlusion CBF, showing sharp rises for CBF < 40%, i.e. the penumbra threshold. This rat model may be useful in providing in vitro reference for studies aiming to validate novel imaging tracers of SNL and MA in vivo.
A new model of transient focal cerebral ischemia for inducing selective neuronal necrosis
2009, Brain Research Bulletin
Brief cerebral ischemia leads to selective neuronal necrosis (SNN), which is characterized by neuronal death with sparing of glial and vascular elements of the central nervous system. Understanding the pathophysiology of SNN may help elucidating the mechanisms and consequences of neuronal injury in humans following brief ischemia. Contrary to the presence of reproducible models of transient global ischemia, animal models of transient focal ischemia producing SNN are scarce and have important limitations such as causing ischemia in a vast area and inducing additional insults. In this study, we developed a practical mouse model of SNN without these limitations, by compressing the distal middle cerebral artery (MCA) with a blunted micropipette for 15 min. The success of compression was evaluated by monitoring the regional cerebral blood flow, and conventional histopathology and immunolabeling of the brain sections. Seven/fourteen days after ischemia, intracisternally administered propidium iodide labeled numerous necrotic cells in the frontoparietal cortex, which were mostly NeuN-positive, but were not immunolabeled with astrocytic markers (GFAP and S100), and showed neuronal morphology with hematoxylin–eosin staining, indicating that the model successfully induced ischemic injury limited to neurons. The model could become an important tool for investigating the long-term effects of brief ischemic events like transient ischemic attacks and could offer convenient reversible distal MCA occlusion for studies using intravital microscopy.

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Ischemia-induced slowly progressive neuronal damage in the rat brain

Abstract

J. Neurol. Sci.

Trends pharmac. Sci.

Brain Res.

Neurosci. Lett.

Neurosci. Lett.

Brain Res.

Brain Res.

Neurosci. Lett.

Brain Res.

Neurosci. Lett.

Prog. Brain Res.

Selective neuronal death after transient forebrain ischemia in the mongolian gerbil: a silver impregnation study

Neuroscience