Elsevier

Brain Research

Volume 1343, 9 July 2010, Pages 143-152
Brain Research

Research Report
Temporal and spatial differences of multiple protein expression in the ischemic penumbra after transient MCAO in rats

https://doi.org/10.1016/j.brainres.2010.04.027Get rights and content

Abstract

Temporal and spatial differences and relationships of proteins relating to the ischemic penumbra were examined at 1, 3, 12, 24, and 48 h after 90 min of transient middle cerebral artery occlusion (tMCAO) in rats. 2, 3, 5-triphenyltetrazolium chloride (TTC) staining showed that the apparent infarction focus first appeared at 1 h after tMCAO, which then largely matured at 24 h. Immunohistochemistry and Western blot indicated no or trace levels of c-fos, hypoxia inducible factor-1α (HIF-1α), heat shock protein 70 (HSP70), and annexin V (A5) positive cells in the sham control brain. Expression of c-fos increased quickly and widely within and outside of the affected arterial territory (peak at 1 h), and that of HIF-1α reached the maximum at 12 h in a smaller area than c-fos. HSP70 began to be induced during the first few hours after tMCAO, peaked at 24 h, then decreased within 48 h, while A5 was slightly expressed at 3 h, then gradually increased until 48 h. Double immunofluorescent analyses showed that the colocalization rates of c-fos/HIF-1α, HIF-1α/HSP70, HSP70/A5, and A5/TUNEL were 40.6%, 58.4%, 42.1% and 61.0%, respectively. These data suggest that multiple molecular penumbra exist after 90 min of tMCAO in the rat brain where several different proteins participate in different temporal and spatial expression patterns. Thus, there is a window for rescue of ischemic neural cells from 12 to 48 h after injury.

Introduction

The ischemic penumbra was first proposed by Astrup et al. (1977), and represents part of the hypoperfused region associated with focal brain ischemia. Additional definitions for the ischemic penumbra have since been proposed from a metabolic view-point (Hossmann, 1994). More recently, the ischemic penumbra has been further defined by characterizing its molecular expression pattern (Sharp et al., 2000). The concept of the multiple molecular penumbra has provided a novel perspective for the study of mechanisms of ischemic brain damage and the monitoring of the damage process, and may allow the development of novel treatment strategies.

The c-fos gene is a key member of the immediate early gene (IEG) family (Funahashi et al., 1999), and is induced in conditions such as pain, epilepsy, cerebral ischemia, and Alzheimer's disease (Fitzgerald, 1990, Gildas, 1988, Hsu et al., 1993, Zhang et al., 1992). Hypoxia inducible factor-1 (HIF-1) is a transcription factor induced by hypoxia (Semenza and Wang, 1992). HIF-1 consists of α and β subunits, and mainly exists as heterodimers of which HIF-1α plays a key role in the promotion of anaerobic metabolism, angiogenesis, and blood vessel dilatation (Ebert et al., 1995, Semenza, 1998). The heat shock protein (HSP) 70 family is a major molecular chaperon and includes two members: constitutive heat shock cognate protein (HSC) 70 and inducible HSP70 (Abe et al., 1993). HSP70 plays the most important role under stressful conditions such as heat shock, hypoxia, cerebral ischemia, and metal intoxication, where HSP70 is induced to prevent intracellular aggregation of denatured proteins for acceleration of the recovery of brain cells (Abe et al., 1991, Abe et al., 1993). Although annexin V (A5) was originally found in the human placenta (Andree et al., 1992), more recent studies suggest that A5 detects cells in the early apoptotic state when the phosphatidylserine (PS) becomes exposed on the surface of the apoptotic cells (Kubista et al., 1999, Narula & Strauss, 2003). Apoptosis is a major cell death pathway activated after cerebral ischemia/reperfusion (Mattson et al., 2001). The terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) reaction can be used to identify cells in the last phase of apoptosis undergoing DNA degradation.

The various proteins described above are important components of the molecular penumbra. However, the relationships between these proteins are largely unknown with respect to temporal and spatial expression. In particular, the exact relationship between A5 and TUNEL is unknown, including time dependent changes of double positive cells after transient middle cerebral artery occlusion (tMCAO). Thus, in the present study we examined the temporal and spatial changes of the molecular penumbra proteins after tMCAO in rats.

Section snippets

Analysis of infarct issue area after transient MCAO

As shown in TTC-stained brain sections, rats subjected to 90 min of tMCAO followed by reperfusion showed development of delayed cerebral infarction (Fig. 1). Sham control rats did not show any lesions in either hemisphere. At 1 h after tMCAO, only a slight infarct was observed in the dorsolateral caudoputamen area, which then progressively developed in the caudoputamen and the cerebral cortex at 3 h and 12 h after tMCAO. Severe cortical and striatal infarcts appeared at 24 h after tMCAO, which

Discussion

The definition of the ischemic penumbra (IP) was recently extended as different genes were reported to be expressed in different zones of spreading depression, hypoxia, denatured protein, and selective neuron death between normal regions and infarction center at different times (termed multiple molecular penumbra) (Sharp et al., 2000). In the present study, we demonstrated that cerebral infarction after tMCAO is a progressive dynamic process. The infarction first appeared at 1 h, enlarged at 3–12

Experimental procedures

Experiments were performed on male Wistar rats at 12 weeks of age (body weight 260–280 g; Japan SLC Inc., Shizuoka, Japan). Animals were maintained for at least 7 days before the experiment in a temperature-regulated room (23–25 °C) on a 12 h light/dark cycle. The rats were fasted but allowed free access to water overnight before surgery.

Acknowledgments

This work was partly supported by Grant-in-Aid for Scientific Research (B) 21390267 and the Ministry of Education, Science, Culture and Sports of Japan, and by Grants-in-Aid from the Research Committee of CNS Degenerative Diseases (Nakano I), and grants (Itoyama Y, Imai T) from the Ministry of Health, Labour and Welfare of Japan.

References (30)

  • Y. Wen et al.

    Estrogen attenuates nuclear factor-kappa B activation induced by transient cerebral ischemia

    Brain Res.

    (2004)
  • P. Zhang et al.

    C-fos protein-like immunoreactivity: distribution in the human brain and over-expression in the hippocampus of patients with Alzheimer's disease

    Neuroscience

    (1992)
  • K. Abe et al.

    Ischemic delayed neuronal death. A mitochondrial hypothesis

    Stroke

    (1995)
  • J. Astrup et al.

    Cortical evoked potential and extracellular K + and H + at critical levels of brain ischemia

    Stroke

    (1977)
  • R.K. Bruick et al.

    A conserved family of prolyl-4-hydroxylases that modify HIF

    Science

    (2001)
  • Cited by (33)

    • Hypoxia-inducible factor-1: Regulatory mechanisms and drug development in stroke

      2021, Pharmacological Research
      Citation Excerpt :

      Except for the area around the infarction, the expression of HIF-1α has also been found in other ischemic regions. In MCAO model, HIF-1α expression was observed in ischemic caudoputamen and cortex, which began to increase at 1 h, peaked at 12 h, and declined at 24 h after reperfusion [17]. In neonatal hypoxia-ischemia model, the expression of HIF-1α increased at 4 h, peaked at 8 h, and declined at 24 h after ischemia in the cortex and hippocampus [18].

    • Morg1<sup>+/-</sup> heterozygous mice are protected from experimentally induced focal cerebral ischemia

      2012, Brain Research
      Citation Excerpt :

      Thus, one may propose a mechanism by which suppression of PHD3 increases HIF-1α in the brain after ischemia leading, via increased HIF-1α with the subsequent induction of various growth factors, to enhanced neuronal recovery. Certainly, the timing of HIF-1α expression may be important (Yeh et al., 2011; Zhang et al., 2010). On the other hand, brain ischemia also upregulates Morg1 expression likely in astrocytes in the penumbra, thus increasing PHD3 stability and activity and thereby limiting availability of HIF-1α.

    • Impaired response of hypoxic sensor protein HIF-1α and its downstream proteins in the spinal motor neurons of ALS model mice

      2012, Brain Research
      Citation Excerpt :

      We suggested that this early and progressive flow-metabolism uncoupling could be profoundly involved in the entire disease process as a vascular component of ALS pathology, suggesting a relative hypoxia in the spinal cord (Miyazaki et al., 2011a). In our previous study, HIF-1α was strongly induced in the ischemic core and penumbra with a peak at 2 h after transient middle cerebral artery occlusion (Zhang et al., 2010). The present study demonstrated a progressive increase in HIF-1α protein expression both in the anterior large MNs and surrounding glial cells of Tg mice from 14 W to 18 W (Fig. 1Ag and h, Bu, and Fig. 3).

    • Lutein enhances survival and reduces neuronal damage in a mouse model of ischemic stroke

      2012, Neurobiology of Disease
      Citation Excerpt :

      Further to this observation, we looked into the underlying mechanisms of lutein's neuroprotective effects in cerebral I/R, which has not been studied previously. The majority of cells in ischemic penumbra die by apoptosis after cerebral ischemia (Zhang et al., 2010). Akt and Bcl-2 play important roles in modulating the intracellular apoptotic signaling pathways.

    View all citing articles on Scopus
    View full text