Neuronal damage and gliosis in the somatosensory cortex induced by various durations of transient cerebral ischemia in gerbils

brain research 1510 (2013) 78–88

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Research Report

Neuronal damage and gliosis in the somatosensory cortex induced by various durations of transient cerebral ischemia in gerbils Jae-Chul Leea,1, Ji Hyeon Ahnb,1, Dae Hwan Leeb, Bing Chun Yana, Joon Ha Parka, In Hye Kima, Geum-Sil Choc, Young-Myeong Kimd, Bonghee Leee, Chan Woo Parkf, Jun Hwi Chof,h, Hui Young Leeg,h,nn, Moo-Ho Wona,h,n a

Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea Laboratory of Neuroscience, Department of Physical Therapy, College of Rehabilitation Science, Daegu University, Gyeongsan 712-714, South Korea c Department of Neuroscience, College of Medicine, Korea University, Seoul 136-705, South Korea d Vascular System Research Center and Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea e Department of Anatomy and Cell Biology, Gachon University of Medicine and Science, Incheon 406-799, South Korea f Department of Emergency Medicine, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea g Department of Internal Medicine, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea h Institute of Medical Sciences, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea b

ar t ic l e in f o

abs tra ct

Article history:

Although many studies regarding ischemic brain damage in the gerbil have been reported,

Accepted 8 March 2013

studies on neuronal damage according to various durations of ischemia–reperfusion (I–R)

Available online 23 March 2013

have been limited. In this study, we examined neuronal damage/death and glial changes in

Keywords:

the somatosensory cortex 4 days after 5, 10 and 15 min of transient cerebral ischemia using

Ischemia–reperfusion

the gerbil. To examine neuronal damage, we used Fluoro-Jade B (F-J B, a marker for

Ischemic duration

neuronal degeneration) histofluorescence staining as well as cresyl violet (CV) staining and

Cerebral cortex

neuronal nuclei (NeuN, neuronal marker) immunohistochemistry. In the somatosensory

Delayed neuronal death

cortex, some CV and NeuN positive (þ) neurons were slightly decreased only in layers III

Astrocytes

and VI in the 5 min ischemia-group, and the number of CVþ and NeuNþ neurons were

Microglia

decreased with longer ischemic time. The F-J B histofluorescence staining showed a clear neuronal damage in layers III and VI, and the number of F-J Bþ neurons was increased with time of ischemia–reperfusion: in the 15 min ischemia-group, the number of F-J Bþ neurons was much higher in layer III than in layer VI. In addition, we immunohistochemically examined gliosis of astrocytes and microglia using anti-glial fibrillary acidic protein (GFAP) and anti-ionized calcium-binding adapter molecule 1 (Iba-1) antibody, respectively. In the

n

Corresponding author at: Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea. Fax: þ82 33 256 1614. nnnn Corresponding author at: Department of internal Medicine, School of Medicine, Kangwon National University, Chuncheon 200-701, South Korea. E-mail addresses: [email protected] (H.Y. Lee), [email protected] (M.-H. Won). 1 Both authors contributed equally to this article. 0006-8993/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2013.03.008

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5 min ischemia-group, GFAPþ astrocytes and Iba-1þ microglia were distinctively increased in number, and their immunoreactivity was stronger than that in the sham-group. In the 10 and 15 min ischemia-groups, numbers of GFAPþ and Iba-1þ glial cells were much more increased with time of ischemia–reperfusion; in the 15 min ischemia-group, their distribution patterns of GFAPþ and Iba-1þ glial cells were similar to those in the 10 min ischemiagroup. Our fining indicates that neuronal death/damage and gliosis of astrocytes and microglia were apparently increased with longer time of ischemia–reperfusion. & 2013 Elsevier B.V. All rights reserved.

1.

Introduction

In comparison with other tissues, the brain is very vulnerable to ischemia because of its high metabolic rate, low oxygen stores and an insufficient reserve of high-energy carbohydrates (Sims and Zaidan, 1995). During global cerebral ischemia, the reduction of blood supply to the brain triggers a number of neuro-pathophysiological processes that result in irreversible neuronal damage, and several regions, such as the hippocampus, of the brain are especially sensitive to transient cerebral ischemia (Kirino, 1982). Selective neuronal death has been shown in the human brain after cardiocirculatory arrest and subsequent cardiopulmonary resuscitation, although the most common clinical cause of the selective neuronal death is ischemia and reperfusion injury (Horn and Schlote, 1992; Petito et al., 1987). In a gerbil model of transient cerebral ischemia, sensitive regions in the brain include the cerebral cortex, striatum and CA1 region of the hippocampus (Hwang et al., 2006, 2007, 2008; Lin et al., 1990; Ohk et al., 2012). In the hippocampus, the vulnerability differs from each hippocampal subregion: The CA1 region is the most vulnerable to ischemia, whereas the CA3 region and dentate gyrus are the most resistant to ischemic insults (Schmidt-Kastner and Freund, 1991; Yu et al., 2012). Especially, neuronal death in the CA1 region is called “delayed neuronal death (DND)” due to that it occurs very slowly (Kirino and Sano, 1984). On the other hand, the somatosensory cortex may play a role in neural rehabilitation by influencing motor function in patients with brain lesions (Conforto et al., 2002; Wu et al., 2005). Neuronal damage in the somatosensory cortex, neurons in which are heterogeneous compared to those in the hippocampal CA1 region, is moderate and observed mainly in layers III and VI of the gerbil and rat somatosensory cortices after ischemia–reperfusion (Hwang et al., 2008; Lin et al., 1990). These brain structures play important roles in the control of different types of sensory or motor behavior, therefore, ischemia–reperfusion injury in the structures is a major cause of neurologic abnormalities (Li et al., 2004; Meno et al., 2003). Although deficits in sensorimotor function are common in humans undergoing hypoxic/ischemic episodes, little is known of selective neuronal death/damage in the somatosensory cortex induced by transient ischemia. The Mongolian gerbil has been used as a good animal model to investigate mechanisms of selective neuronal death following transient global cerebral ischemia (Bian et al., 2007; Lorrio et al., 2009; Zhang et al., 2009), because about 90% of gerbils lack the communicating vessels between the carotid and vertebral circulations. Thus, the bilateral occlusion of the

common carotid arteries essentially completely eliminates blood flow to the forebrain while completely sparing the vegetative centers of the brain stem. Although there has been a great deal of information on ischemic damage in the gerbil brain (Fukuchi et al., 1998; Janac et al., 2006), studies regarding neuronal damage/death in the somatosensory cortex according to the duration of ischemia–reperfusion have been limited. Therefore, the present study was undertaken in order to identify the degree of neuronal injury after various durations of transient cerebral ischemia using cresyl violet staining, NeuN immunohistochemistry and Fluoro-Jade B (F-J B) histofluorescence. F-J B is a very useful maker for neuronal degeneration (Schmued and Hopkins, 2000). In addition, we examined changes of astrocytes and microglia in the gerbil somatosensory cortex after ischemia–reperfusion.

2.

Results

2.1.

Spontaneous locomotor activity

To investigate change in motor behavior after ischemic damage, spontaneous motor activity was examined 24 h after ischemia–reperfusion in all the groups (Fig. 1). The spontaneous locomotor activity was significantly increased in the 5 min ischemia-group (mean7S.E.M., 570761) after ischemia–reperfusion compared to that in the sham-group (298748). In the 10 min and 15 ischemia-group, the activity was more increased (760754 vs 1100740) than the 5 min ischemia-group.

Fig. 1 – Locomotor activity of the sham- and ischemiagroups (5, 10 and 15 min). The spontaneous locomotor activity is evaluated in terms of entire distance (meters) traveled for 60 min (n ¼ 10 per group; þPo0.05, significantly different from the sham group; #Po0.05, significantly different from the preceding-group). The bars indicate the means7SEM.

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2.2.

Neuronal damage

2.2.1.

CV positive cells

We used CV staining to examine neuronal damage in the gerbil somatosensory cortex. In the present study, neurons were identified morphologically by their larger, pale nuclei surrounded by darkly stained cytoplasm containing Nissl bodies (diameter 410 mm). In the sham-group, neurons in the somatosensory cortex were well stained with CV (Fig. 2A, a, b and Table 1). In the 5 min ischemia-group, we found that CV positive neurons showed a tendency to be decreased in layer III and in the upper part of layer VI at 4 days after ischemia–perfusion (Fig. 2B, c d and Table 1). In the 10 min and 15 min ischemia-groups, CV positive neurons were dramatically decreased in layers III compared to that in the 5 min ischemia-group (Fig. 2C, D, e–h and Table 1).

2.2.2.

NeuN immunoreactive cells

In the sham-group, neurons in the somatosensory cortex were well stained with NeuN (Fig. 3A, a, b and Table 1). In the 5 min ischemia-group, NeuN immunoreactive neurons were slightly decreased at 4 days post-ischemia, and this was accompanied by a reduction in the number of CV positive

neurons in layer III and in the upper part of layer VI (Fig. 3B, c, d and Table 1). NeuN immunoreactive neurons were decreased with time after ischemia–reperfusion. In the 10 min and 15 min ischemia-groups, Numbers of NeuN immunoreactive neurons were dramatically decreased in layers III and VI compared to those in the 5 min ischemiagroup (Figs. 3C, D, e–h and Table 1).

2.2.3.

F-J B positive cells

Neuronal degeneration in the somatosensory cortex following ischemia–reperfusion was examined using the F-J B histofluorescence staining. In the sham-group, no F-J B positive neurons were observed in the somatosensory cortex (Fig. 4A, a, b and Table 1). Four days after ischemia–reperfusion in the 5 min ischemia-group, some F-J B positive neurons were observed in layer III and in the upper part of layer VI; in layers I, II, IV and V, we could not find F-J B positive neurons (Fig. 4B, c, d and Table 1). In the 10 min ischemia-group, the mean number of F-J B positive neurons were dramatically increased in layer III and in the upper part of layer VI compared to those in the 5 min ischemia-group (Figs. 4C, e, f and Table 1). In the 15 min ischemia-group, many F-J B positive cells were also detected in layers III and VI (Fig. 4D, g, h and Table 1).

Fig. 2 – CV staining of the somatosensory cortex of the sham-group (A, a and b) and ischemia-groups (5 min, B, c, and d; 10 min, C, e and f; 15 min, D, g and h) 4 days after ischemia–reperfusion. In the sham-group, CV positive neurons are distributed in all the layers of the somatosensory cortex. In all the ischemia-groups, numbers of CV positive neurons are decreased with time in layer III (upper squares; c, e and g) and in the upper part of layer VI (below squares; d, f and h) of the somatosensory cortex. Scale bar¼ 200 lm (A–D), 30 lm (a–h).

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Table 1 – The number of cresyl violet, NeuN and F-J B positive neurons/40 mm2 in a section of the somatosensory cortex 4 days after ischemia–reperfusion in sham- and ischemia-groups. Duration of ischemia–reperfusion Sham

5 min

10 min

15 min

Cresyl violet

Layer III Layer VI

132.077.0 120.079.0

116.0711.0* 102.0710.0*

81.079.0*,** 85.078.0*

75.0710.0* 70.0712.0*

NeuN

Layer III Layer VI

120.077.5 108.078.0

104.078.0* 90.078.6*

72.0711.0*,** 74.079.0*

63.0710.0*,** 55.077.0*,**

F-J B

Layer III Layer VI

0.370.2 0.470.1

10.072.0* 9.071.5*

19.072.5*,** 14.072.0*

35.073.0*,** 20.073.5*,**

n ¼ 10 per group. n Po0.05, significantly different from the sham-group. nn Po0.05, significantly different from the preceding-group. Data are defined as the means7SEM.

Fig. 3 – NeuN immunohistochemistry in the somatosensory cortex of the sham-group (A, a and b) and ischemia-groups (5 min, B, c, and d; 10 min, C, e and f; 15 min, D, g and h) 4 days after ischemia–reperfusion. In the sham-group, NeuNimmunoreactive neurons are distributed in all the layers of the somatosensory cortex (A, a and b). In all the ischemiagroups, NeuN-immunoreactive neurons are decreased in layer III (upper squares; c, e and g) and in the upper part of layer VI (below squares; d, f and h) of the somatosensory cortex. Scale bar¼200 lm (A–D), 30 lm (a–h).

2.3.

GFAP immunoreactive astrocytes

In the somatosensory cortex of the sham-group, GFAP immunoreactive astrocytes showed a resting form with a

small cell body and thin processes (Fig. 5A, a, b, Table 2). In the 5 min ischemia-group, GFAP immunoreactive astrocytes were increased in layer III and in the upper part of layer VI compared to those in the sham-group (Fig. 5B, c, d, Table 2).

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In the 10 min ischemia-group, the immunoreactivity of GFAP immunoreactive astrocytes were apparently increased in layer III and in the upper part of layer VI compared to those in the 5 min ischemia-group (Fig. 5C, e, f, Table 2); however, they were not significantly changed in the 15 min ischemiagroup compared to those in the 10 min ischemia-group (Fig. 5D, g, h, Table 2).

2.4.

Iba-1-immunoreactive microglia

In the present study, we found that Iba-1 immunoreactive microglia showed a resting form in the sham-group (Fig. 6A, a, b, Table 2). Iba-1 immunoreactive microglia were increased in the 5 min ischemia-group (Figs. 6B, c, d, Table 2). In the 10 min ischemia-group, the number of Iba-1 immunoreactive microglia was dramatically increased, and their size was much larger than that in the sham-group (Fig. 6C, e, f, Table 2). In the 15 min ischemia-group, the immunoreactivity of Iba-1 positive microglia was also increased in layer III and in the upper part of layer VI compared to those in the 10 min ischemia-group (Fig. 6D, g, h, Table 2).

3.

Discussion

The somatosensory cortex in the brain is very important to control complex senses and movements (Gentilucci et al., 1997) and a somatosensory stimulation improves neural rehabilitation in patients with brain lesions (Conforto et al., 2002). Pyramidal neurons constitute major cells in the somatosensory cortex, and the projection target of these cells generally depends on the layer in which their soma resides (Markram et al., 1997). They are thought to be fundamental for cortical tasks such as feature selection and perceptual grouping (Grossberg and Raizada, 2000). Their morphology is similar to neurons in layer VI, but their apical dendrites are thinner and shorter (Larkman, 1991). Why the pyramidal cells in layer III and the small pyramidal cells in layer VI should be so much more sensitive to ischemia than other cortical neurons has been unknown. This phenomenon may be related to the specificity of local vascular supply and the immaturity of penetrator vessels from superficial arteries (Rorke, 1992). Moreover, many pyramidal cells in layer III are corticocortical projection cells (Chapin and Lin, 1984) and

Fig. 4 – F-J B staining of the somatosensory cortex in the sham-group (A, a and b) and ischemia-groups (5 min, B, c, and d; 10 min, C, e and f; 15 min, D, g and h) 4 days after ischemia–reperfusion. In the sham-group, No F-J B positive neurons are observed in all the layers of the somatosensory cortex. In all the ischemia-groups, numbers of F-J B positive neurons are increased with time in layer III (upper squares; c, e and g) and in the upper part of layer VI (below squares; d, f and h) of the somatosensory cortex. Scale bar¼ 200 lm (A–D), 30 lm (a–h).

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Fig. 5 – GFAP immunohistochemistry in the somatosensory cortex of the sham-group (A, a and b) and ischemia-groups (5 min, B, c, and d; 10 min, C, e and f; 15 min, D, g and h) 4 days after ischemia–reperfusion. In all the ischemia-groups, GFAP immunoreactive astrocytes are increased in number and immunoreactivity in layer III (upper squares; c, e and g) and in the upper part of layer VI (below squares; d, f and h) of the somatosensory cortex compared to those in the sham-group. Scale bar¼ 200 lm (A–D), 30 lm (a–h).

Table 2 – The number of GFAP and Iba-1 positive astrocyte or microglia/40 mm2 in a section of the somatosensory cortex 4 days after ischemia–reperfusion in sham- and ischemia-groups. Duration of ischemia–reperfusion Sham

5 min

10 min

15 min

GFAP

Layer III Layer VI

8.573.0 9.072.7

20.073.0* 17.472.0*

41.274.4*,** 37.072.8*,**

36.075.0* 39.074.2*

Iba-1

Layer III Layer VI

8.071.5 9.272.0

12.072.8* 18.073.4*

25.074.2*,** 28.374.0*,**

39.573.4*,** 27.074.0*,**

n¼ 10 per group. n Po0.05, significantly different from the sham-group. nn Po0.05, significantly different from the preceding-group. Data are defined as the means7SEM.

some neurons in layer VI are callosal projection cells (Olavarria et al., 1984), therefore, there is a close relationship between motor activity and the responsiveness of somatosensory neurons in freely moving animals (Chapin and Lin, 1984). In addition, many studies have described that transient cerebral ischemia led to locomotor hyperactivity (Gerhardt and Boast, 1988; Karasawa et al., 1994; Kuroiwa et al., 1991). In the presents study, we also found that spontaneous motor

activity was highest 1 day after ischemia–reperfusion, and the activity was highest in the 15 min ischemia-group. Therefore, the increased locomotor activity in the gerbil after ischemia–reperfusion could also be explained as a transient response to cortical degeneration. In present study, we found a significant loss of CV positive cells and NeuN-immunoreactive neurons in both layer III and the upper part of layer V of the ischemic somatosensory

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Fig. 6 – Immunohistochemistry for Iba-1 in the somatosensory cortex of the sham-group (A, a and b) and ischemia-groups (5 min, B, c, and d; 10 min, C, e and f; 15 min, D, g and h) 4 days after ischemia–reperfusion. In all the ischemia-groups, Iba-1 immunoreactive microglia are increased in number and immunoreactivity in both layer III (upper squares; c, e and g) and the upper part of layer VI (below squares; d, f and h) of the somatosensory cortex compared to those in the sham-group. Scale bar¼ 200 lm (A–D), 30 lm (a–h).

cortex region 4 days after ischemia–reperfusion. CV has been widely used for a histological method to identify cell damage in the nervous system, because it binds to acidic components in the cytoplasm (Alvarez-Buylla et al., 1990). Damaged cells show various features including a shrunken cell body with pyknosis and chromatolysis (Bartus et al., 1995). However, this method is insufficient to discriminate neuronal degeneration, because the presence of argyrophilic dark neurons simply reflects exposure to an insult that would ultimately result in the neurons either dying or recovering (Gallyas et al., 1992). On the other hand, it is important to count neuronal, not glial, loss in a damaged brain, because NeuN immunohistochemistry shows an apparent neuronal loss. In addition, we used F-J B histofluorescence to elucidate the degree of neuronal damage in the somatosensory cortex of the gerbil brain after various durations of ischemia. F-J B has a good affinity for entirely degenerating neurons (cell bodies, dendrites, axons and axon terminals), and it is a useful marker for study on neuronal degeneration after ischemic injury (Schmued and Hopkins, 2000). On the other hand, in the present study, glia-like cells were excluded from counts by their relatively smaller size and lack of stained cytoplasm (diameter o10 mm, data not

shown) in the CV stained sections. These small CV-positive cells showed a tendency to be increased in layer III and in the upper part of layer VI 4 days after ischemia–reperfusion compared to the sham-group. Therefore, we compared the changes of astrocytes and microglia in the ischemic somatosensory cortex region according to various durations of ischemia–reperfusion. It is well established that hippocampal CA1 pyramidal neurons are vulnerable to ischemic damage. Kirino and Sano (1984) showed that ischemia for 5 min in the gerbil brain produced pure pyramidal cell death in the CA1 region; no definite cell injury was not found in the CA2 and CA3 regions after 5 min of ischemia. However, the authors found that, with longer ischemia (20–30 min), pyramidal neurons in the CA2 and CA3 regions showed reactive changes. However, we recently reported that degenerating CA1 pyramidal neurons were found in the 5 min, 10 min, 15 min and 20 min ischemia-groups; most of the CA1 pyramidal neurons of these groups were almost completely degenerated in the brain induce by at least 5 min of transient cerebral ischemia (Yu et al., 2012). On the other hand, Ito et al. (1975) showed that neuronal damage was examined using a light microscopic observation in the gerbil striatum after occlusion of 15 min.

brain research 1510 (2013) 78–88

However, we recently found that degenerating neurons in the gerbil striatum were rarely detected in the 15 min ischemiagroup using the F-J B histofluorescence staining (Ohk et al., 2012). In this study, we observed that F-J Bþ cells were detected in the somatosensory cortex 4 days after ischemia–reperfusion. A few pyramidal neurons in layers III and in the upper part of layer VI were positive to F-J B in the 5 min ischemia-group, and the number of F-B positive neurons was increased with longer ischemic duration of duration, showing that many neurons in layer III were positive to F-J B in the 15 min ischemia-group. This result is supported by our previous study in which argyrophilic neurons appeared in layers III and VI of the gerbil somatosensory cortex after 5 min of ischemia (Hwang et al., 2008). In addition, Lin et al. (1990) reported that neurons in layers III and VI were sensitive to ischemic damage in gerbils; neurons in layers II, IV and V were relatively resistant to ischemic insult. Markers for astrocytes (Petito et al., 1990) and microglia (Giordana et al., 1994) provide useful indices of glial responses to transient ischemia. It is well known that, in the hippocampus, neuronal death/damage is accompanied by the activation of astrocytes and microglia, and that the activation of these cells releases a variety of cytotoxic agents that lead to neuronal injury (Giulian and Vaca, 1993). Our result showed reactive astrogliosis in the gerbil somatosensory cortex 4 days after ischemia–reperfusion: GFAP immunoreactivity was increased with longer ischemic time; however, the immunoreactivity in the 15 min ischemia-group was similar to that in the 10 min ischemia-group. The increased expression of GFAP, which shows the main constituent of intermediate filaments in astrocytes, is a hallmark of reactive astrogliosis (Petito et al., 1990). The reactive astrogliosis occurs prominently in response to ischemic insults; however, the functions of reactive astrogliosis are not well understood yet. It was reported that GFAP-up-regulated astrocytes were able to uptake harmful substances such as excitatory neurotransmitters and could produce neurotrophic factors under pathological conditions (Kraig et al., 1991; Lascola and Kraig, 1997; Matsushima et al., 1998). In the present study, many GFAP-immunoreactive astrocytes were aggregated in layer III and in the upper part of layer VI 4 days after ischemia– reperfusion compared to those in the sham-group. Our present finding suggests that the increase of GFAP immunoreactivity in the somatosensory cortex in the 15 min ischemiagroup may be associated with a secretion of harmful substances by transient ischemic damage. Microglia are resident macrophages and distributed throughout the brain parenchyma. Changes in the morphology and function of microglia are closely correlated with the development of delayed neuronal death in ischemia (Hailer et al., 1996; Hwang et al., 2006; Schwartz et al., 2006). Microglia can contribute to the elimination of deleterious debris, the promotion of tissue repair and the return to tissue homeostasis, and they may involve in neuroprotection (Hashimoto et al., 2005; Laurenzi et al., 2001; Lu et al., 2005). On the other hand, microglia secrete large amounts of cytotoxic and inflammatory mediators (Colton and Gilbert, 1987; Han et al., 2002; Suzuki et al., 1999). In addition, microglial activation after ischemic damage leads to neuronal death/ damage through excitotoxic mechanisms (Barron, 1995;

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Kreutzberg, 1996; Lehrmann et al., 1995). We, in the present study, observed that the number and immunoreactivity of Iba-1-immunoreactive microglia were increased with longer time of ischemia. In the 15 min ischemia-group, microglia were larger in size and higher in immunoreactivity than those in the 10 min ischemia-group. Although we could not exactly explain why the activation of microglia was increased in the ischemia-group, it seems that Iba-1-immunoreactive microglia may be closely related to the neuronal degeneration detected by the F-J B staining after ischemic damage. In the present study, the data were made only at 4 days according to the duration time of ischemia–reperfusion. So, further experiments are needed to clarify the data. We previously reported that long-term changes in neuronal degeneration and microglial activation in the gerbil hippocampal CA1 region after 5 min of transient cerebral ischemia using specific markers for neuronal damage and microgliosis (Lee et al., 2010). F-J B positive neurons in the stratum pyramidale of the ischemic CA1 region were shown from 4 days to 45 days after ischemia–reperfusion. In addition, Iba-1 positive microglia were markedly increased after ischemia– reperfusion, and peaked at 15 days after ischemia–reperfusion. Thereafter, Iba-1 immunoreactivity was decreased with time-dependent manner in the ischemic CA1 region. These results indicate that ischemia-induced neuronal degeneration lasts about 45 days in the CA1 region after transient ischemic damage, and microglial activation may be closely related to the neuronal degeneration. In conclusion, we found that neuronal degeneration/death (CV-, NeuN- and F-J B-positive cells) occurred differently in the gerbil somatosensory cortex according to ischemic duration, and that the activations of astrocytes and microglia in the ischemic cerebral cortex were different according to the duration time of transient ischemia. These results indicate that the degree of neuronal death/damage and gliosis in an ischemic brain must be distinctively different according to ischemic duration.

4.

Experimental procedures

4.1.

Experimental animals

Male Mongolian gerbils (Meriones unguiculatus) were obtained from the Experimental Animal Center, Hallym University, Chuncheon, South Korea. Gerbils were used at 6 months (B.W., 65–75 g) of age. The animals were housed in a conventional state under adequate temperature (23 1C) and humidity (60%) control with a 12-h light/12-h dark cycle, and were provided with free access to food and water. The procedures for animal handling and care adhered to guidelines that are in compliance with the current international laws and policies (Guide for the Care and Use of Laboratory Animals, The National Academies Press, 8th Ed., 2011), and they were approved by the Institutional Animal Care and Use Committee (IACUC) at Hallym's Medical Center. All of the experiments were conducted to minimize the number of animals used and the suffering caused by the procedures used in the present study.

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4.2.

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Induction of transient cerebral ischemia

The animals were anesthetized with a mixture of 2.5% isoflurane (Baxtor, Deerfield, IL) in 33% oxygen and 67% nitrous oxide. Bilateral common carotid arteries were isolated and occluded using non-traumatic aneurysm clips. The complete interruption of blood flow was confirmed by observing the central artery in retinae using an ophthalmoscope. After 5 min (5 min ischemia group), 10 min (10 min ischemia group) or 15 min (15 min ischemia group) of occlusion, the aneurysm clips were removed from the common carotid arteries. The body (rectal) temperature under free-regulating or normothermic (3770.5 1C) conditions was monitored with a rectal temperature probe (TR-100; Fine Science Tools, Foster City, CA) and maintained using a thermometric blanket before, during and after the surgery until the animals completely recovered from anesthesia. Thereafter, animals were kept on the thermal incubator (Mirae Medical Industry, Seoul, South Korea) to maintain the body temperature of animals until the animals were euthanized. Sham-operated animals (sham-operated group) were subjected to the same surgical procedures except that the common carotid arteries were not occluded.

4.3.

Spontaneous locomotor activity

To elucidate ischemia-induced hyperactivity, spontaneous motor activity was measured according to previous studies (Yan et al., 2011). For spontaneous motor activity, gerbils (n ¼10 in each group) were individually placed in a Plexiglas cage (25 cm  20 cm  12 cm), located inside a soundproof chamber. Locomotor activity was recorded with Photobeam Activity System—Home Cage (San Diego Instruments). The cage was fitted with two parallel horizontal infrared beams 2 cm off the floor. Movement was detected by the interruption of an array of 32 infrared beams produced by photocells. Spontaneous locomotor activity was monitored for 60 min, 1 day after ischemia and, simultaneously, the number of times each animal reared and the time (in seconds) spent in grooming behavior were recorded. Locomotor activity data were acquired by an AMB analyzer (IPC Instruments, Berks, U. K.). Results were evaluated in terms of entire distance (meters) traveled for 60 min test period.

4.4.

Tissue processing for histology

For histology, sham- and ischemia-operated gerbils (shamand ischemia-groups) (n¼ 10 in each group) were sacrificed 4 days after ischemia–reperfusion. Animals were anesthetized with pentobarbital sodium and perfused transcardially with 0.1 M phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in 0.1 M phosphate-buffer (PB, pH 7.4). The brains were removed and postfixed in the same fixative for 6 h. The brain tissues were cryoprotected by infiltration with 30% sucrose overnight. Thereafter, frozen tissues were serially sectioned on a cryostat (Leica, Germany) into 30-μm coronal sections, and they were then collected into six-well plates containing PBS.

4.5.

Cresyl violet (CV) staining

To examine the neuronal death in the brain after transient cerebral ischemia, sham- and ischemia-groups, the sections were mounted on gelatin-coated microscopy slides. Cresyl violet acetate (Sigma, MO, USA) was dissolved at 1.0% (w/v) in distilled water, and glacial acetic acid was added to this solution. the sections were stained and dehydrated by immersing in serial ethanol baths, and they were then mounted with Canada balsam (Kato, Japan).

4.6.

F-J B histofluorescence

To confirm the neuronal death in the brain after transient forebrain ischemia, sham- and ischemia-operated animals (n ¼10 in each group) were used 4 days after the ischemic surgery for F-J B (a high affinity fluorescent marker for the localization of neuronal degeneration) histofluorescence under the same conditions. F-J B histofluorescence staining procedures were conducted according to the method by Candelario-Jalil et al. (2003). The sections were first immersed in a solution containing 1% sodium hydroxide in 80% alcohol, and followed in 70% alcohol. They were then transferred to a solution of 0.06% potassium permanganate, and transferred to a 0.0004% F-J B (Histochem, Jefferson, AR, USA) staining solution. After washing, the sections were placed on a slide warmer (approximately 50 1C), and then examined using an epifluorescent microscope (Carl Zeiss, Germany) with blue (450–490 nm) excitation light and a barrier filter. With this method neurons that undergo degeneration brightly fluoresce in comparison to the background (Schmued and Hopkins, 2000).

4.7.

Immunohistochemistry for NeuN, GFAP and Iba-1

In order to examine the changes of neurons, astrocytes and microglia in the somatosensory cortex after ischemia–reperfusion, we carried out immunohistochemical staining with mouse anti-neuronal nuclei (NeuN; 1:1000, Chemicon International, Temecula, CA) for neurons, rabbit anti-glial fibrillary acidic protein (GFAP, 1:800, Chemicon) for astrocytes, rabbit anti-ionized calcium-binding adapter molecule 1 (Iba-1, 1:500, Wako, Japan) for microglia. In brief, the sections were sequentially treated with 0.3% hydrogen peroxide (H2O2) in PBS for 30 min and 10% normal goat serum in 0.05 M PBS for 30 min. The sections were next incubated with anti-NeuN, anti-GFAP or Iba-1 overnight at 4 1C. Thereafter the tissues were exposed to biotinylated goat anti-mouse or -rabbit IgG (Vector, Burlingame, CA) and streptavidin peroxidase complex (1:200, Vector). And they were visualized by staining with 3,3′-diaminobenzidine tetrahydrochloride in 0.1 M Tris–HCl buffer (pH 7.2) and mounted on gelatin-coated slides. After dehydration, the sections were mounted with Canada balsam (Kanto, Tokyo, Japan).

4.8.

Cell counts

All measurements were performed to insure objectivity in blind conditions, by two observers for each experiment, carrying out the measures of experimental samples under

brain research 1510 (2013) 78–88

the same conditions. The studied tissue sections were selected with 120 mm interval according to anatomical landmarks corresponding to AP from þ0.3 to −1.3 mm of gerbil brain atlas (Loskota and Verity, 1974), and cell counts were obtained by averaging the counts from 25 sections taken from each animal. Digital images of the somatosensory cortex were captured with an AxioM1 light microscope (Carl Zeiss, Germany) equipped with a digital camera (Axiocam, Carl Zeiss, Germany) connected to a PC monitor. The number of CV, NeuN, F-J B, GFAP and Iba-1 positive cells was counted in a 200  200 mm2 square applied approximately at layers III and VI of the somatosensory cortex region. Cell counts were obtained by averaging the total cell numbers from each animal per group. Cell quantification was performed in the right and left side, respectively, in each animal. However, we did not find any statistical difference between both sides. We calculated a correction factor using Abercrombie's formula, because profile counts could produce some overcount depending on the thickness and height of the counted object (Guillery, 2002). The actual calculated number of cells (N) was obtained using the following formula: N¼ n  (T/(Tþh)). The zaxis was calculated by measuring the focus changes in the camera and calibrated by the software in reference to a coverslip of 0.22-mm thickness.

4.9.

Statistical analysis

Data are expressed as the mean7SEM. The data were evaluated by a Tukey test for post-hoc multiple comparisons following one-way ANOVA. Statistical significance was considered at Po0.05.

Acknowledgments The authors would like to thank Mr. Seung Uk Lee for their technical help in this study. This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (20100010580), and by a grant from the National R&D Program for Cancer Control, Ministry for Health and Welfare, Republic of Korea (1020420).

r e f e r e n c e s

Alvarez-Buylla, A., Ling, C.Y., Kirn, J.R., 1990. Cresyl violet: a red fluorescent Nissl stain. J. Neurosci. Methods 33, 129–133. Barron, K.D., 1995. The microglial cell. A historical review. J. Neurol. Sci. 134 (Suppl.), 57–68. Bartus, R.T., Dean, R.L., Cavanaugh, K., Eveleth, D., Carriero, D.L., Lynch, G., 1995. Time-related neuronal changes following middle cerebral artery occlusion: implications for therapeutic intervention and the role of calpain. J. Cereb. Blood Flow Metab. 15, 969–979. Bian, Q., Shi, T., Chuang, D.M., Qian, Y., 2007. Lithium reduces ischemia-induced hippocampal CA1 damage and behavioral deficits in gerbils. Brain Res. 1184, 270–276. Candelario-Jalil, E., Alvarez, D., Merino, N., Leon, O.S., 2003. Delayed treatment with nimesulide reduces measures of oxidative stress following global ischemic brain injury in gerbils. Neurosci. Res. 47, 245–253.

87

Chapin, J.K., Lin, C.S., 1984. Mapping the body representation in the SI cortex of anesthetized and awake rats. J. Comp. Neurol. 229, 199–213. Colton, C.A., Gilbert, D.L., 1987. Production of superoxide anions by a CNS macrophage, the microglia. FEBS Lett. 223, 284–288. Conforto, A.B., Kaelin-Lang, A., Cohen, L.G., 2002. Increase in hand muscle strength of stroke patients after somatosensory stimulation. Ann. Neurol. 51, 122–125. Fukuchi, T., Katayama, Y., Kamiya, T., McKee, A., Kashiwagi, F., Terashi, A., 1998. The effect of duration of cerebral ischemia on brain pyruvate dehydrogenase activity, energy metabolites, and blood flow during reperfusion in gerbil brain. Brain Res. 792, 59–65. Gallyas, F., Zoltay, G., Dames, W., 1992. Formation of “dark” (argyrophilic) neurons of various origin proceeds with a common mechanism of biophysical nature (a novel hypothesis). Acta Neuropathol. 83, 504–509. Gentilucci, M., Toni, I., Daprati, E., Gangitano, M., 1997. Tactile input of the hand and the control of reaching to grasp movements. Exp. Brain Res. 114, 130–137. Gerhardt, S.C., Boast, C.A., 1988. Motor activity changes following cerebral ischemia in gerbils are correlated with the degree of neuronal degeneration in hippocampus. Behav. Neurosci. 102 (301-3), 328. Giordana, M.T., Attanasio, A., Cavalla, P., Migheli, A., Vigliani, M. C., Schiffer, D., 1994. Reactive cell proliferation and microglia following injury to the rat brain. Neuropathol. Appl. Neurobiol. 20, 163–174. Giulian, D., Vaca, K., 1993. Inflammatory glia mediate delayed neuronal damage after ischemia in the central nervous system. Stroke 24, I84–I90. Grossberg, S., Raizada, R.D., 2000. Contrast-sensitive perceptual grouping and object-based attention in the laminar circuits of primary visual cortex. Vision Res. 40, 1413–1432. Guillery, R.W., 2002. On counting and counting errors. J. Comp. Neurol. 447, 1–7. Hailer, N.P., Jarhult, J.D., Nitsch, R., 1996. Resting microglial cells in vitro: analysis of morphology and adhesion molecule expression in organotypic hippocampal slice cultures. Glia 18, 319–331. Han, H.S., Qiao, Y., Karabiyikoglu, M., Giffard, R.G., Yenari, M.A., 2002. Influence of mild hypothermia on inducible nitric oxide synthase expression and reactive nitrogen production in experimental stroke and inflammation. J. Neurosci. 22, 3921–3928. Hashimoto, M., Nitta, A., Fukumitsu, H., Nomoto, H., Shen, L., Furukawa, S., 2005. Involvement of glial cell line-derived neurotrophic factor in activation processes of rodent macrophages. J. Neurosci. Res. 79, 476–487. Horn, M., Schlote, W., 1992. Delayed neuronal death and delayed neuronal recovery in the human brain following global ischemia. Acta Neuropathol. 85, 79–87. Hwang, I.K., Yoo, K.Y., Kim, D.W., Choi, S.Y., Kang, T.C., Kim, Y.S., Won, M.H., 2006. Ionized calcium-binding adapter molecule 1 immunoreactive cells change in the gerbil hippocampal CA1 region after ischemia/reperfusion. Neurochem. Res. 31, 957–965. Hwang, I.K., Yoo, K.Y., Kim do, H., Lee, B.H., Kwon, Y.G., Won, M. H., 2007. Time course of changes in pyridoxal 5′-phosphate (vitamin B6 active form) and its neuroprotection in experimental ischemic damage. Exp. Neurol. 206, 114–125. Hwang, I.K., Yoo, K.Y., Kim, D.W., Kwon, O.S., Lim, S.S., Kang, I.J., Choi, S.Y., Won, M.H., 2008. Differential changes in pyridoxine 5′-phosphate oxidase immunoreactivity and protein levels in the somatosensory cortex and striatum of the ischemic gerbil brain. Neurochem. Res. 33, 1356–1364.

88

brain research 1510 (2013) 78–88

Ito, U., Spatz, M., Walker Jr., J.T., Klatzo, I., 1975. Experimental cerebral ischemia in mongolian gerbils. I. Light microscopic observations. Acta Neuropathol. 32, 209–223. Janac, B., Radenovic, L., Selakovic, V., Prolic, Z., 2006. Time course of motor behavior changes in Mongolian gerbils submitted to different durations of cerebral ischemia. Behav. Brain Res. 175, 362–373. Karasawa, Y., Araki, H., Otomo, S., 1994. Changes in locomotor activity and passive avoidance task performance induced by cerebral ischemia in Mongolian gerbils. Stroke 25, 645–650. Kirino, T., 1982. Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res. 239, 57–69. Kirino, T., Sano, K., 1984. Selective vulnerability in the gerbil hippocampus following transient ischemia. Acta Neuropathol. 62, 201–208. Kraig, R.P., Dong, L.M., Thisted, R., Jaeger, C.B., 1991. Spreading depression increases immunohistochemical staining of glial fibrillary acidic protein. J. Neurosci. 11, 2187–2198. Kreutzberg, G.W., 1996. Microglia: a sensor for pathological events in the CNS. Trends Neurosci. 19, 312–318. Kuroiwa, T., Bonnekoh, P., Hossmann, K.A., 1991. Locomotor hyperactivity and hippocampal CA1 injury after transient forebrain ischemia of gerbils. Neurosci. Lett. 122, 141–144. Larkman, A.U., 1991. Dendritic morphology of pyramidal neurones of the visual cortex of the rat: I. Branching patterns. J. Comp. Neurol. 306, 307–319. Lascola, C., Kraig, R.P., 1997. Astroglial acid–base dynamics in hyperglycemic and normoglycemic global ischemia. Neurosci. Biobehav. Rev. 21, 143–150. Laurenzi, M.A., Arcuri, C., Rossi, R., Marconi, P., Bocchini, V., 2001. Effects of microenvironment on morphology and function of the microglial cell line BV-2. Neurochem. Res. 26, 1209–1216. Lee, C.H., Yoo, K.Y., Choi, J.H., Park, O.K., Hwang, I.K., Kim, S.K., Kang, I.J., Kim, Y.M., Won, M.H., 2010. Neuronal damage is much delayed and microgliosis is more severe in the aged hippocampus induced by transient cerebral ischemia compared to the adult hippocampus. J. Neurol. Sci. 294, 1–6. Lehrmann, E., Kiefer, R., Finsen, B., Diemer, N.H., Zimmer, J., Hartung, H.P., 1995. Cytokines in cerebral ischemia: expression of transforming growth factor beta-1 (TGF-beta 1) mRNA in the postischemic adult rat hippocampus. Exp. Neurol. 131, 114–123. Li, X., Blizzard, K.K., Zeng, Z., DeVries, A.C., Hurn, P.D., McCullough, L.D., 2004. Chronic behavioral testing after focal ischemia in the mouse: functional recovery and the effects of gender. Exp. Neurol. 187, 94–104. Lin, C.S., Polsky, K., Nadler, J.V., Crain, B.J., 1990. Selective neocortical and thalamic cell death in the gerbil after transient ischemia. Neuroscience 35, 289–299. Lorrio, S., Negredo, P., Roda, J.M., Garcia, A.G., Lopez, M.G., 2009. Effects of memantine and galantamine given separately or in association, on memory and hippocampal neuronal loss after transient global cerebral ischemia in gerbils. Brain Res. 1254, 128–137. Loskota, William James, Lomax, Peter, Anthony Verity, M. (Eds.), 1974. Ann Arbor Science, Ann Arbor. Lu, Y.Z., Lin, C.H., Cheng, F.C., Hsueh, C.M., 2005. Molecular mechanisms responsible for microglia-derived protection of Sprague-Dawley rat brain cells during in vitro ischemia. Neurosci. Lett. 373, 159–164. Markram, H., Lubke, J., Frotscher, M., Roth, A., Sakmann, B., 1997. Physiology and anatomy of synaptic connections between

thick tufted pyramidal neurones in the developing rat neocortex. J. Physiol. 500 (Pt 2), 409–440. Matsushima, K., Schmidt-Kastner, R., Hogan, M.J., Hakim, A.M., 1998. Cortical spreading depression activates trophic factor expression in neurons and astrocytes and protects against subsequent focal brain ischemia. Brain Res. 807, 47–60. Meno, J.R., Higashi, H., Cambray, A.J., Zhou, J., D’Ambrosio, R., Winn, H.R., 2003. Hippocampal injury and neurobehavioral deficits are improved by PD 81,723 following hyperglycemic cerebral ischemia. Exp. Neurol. 183, 188–196. Ohk, T.G., Yoo, K.Y., Park, S.M., Shin, B.N., Kim, I.H., Park, J.H., Ahn, H.C., Lee, Y.J., Kim, M.J., Kim, T.Y., Won, M.H., Cho, J.H., 2012. Neuronal damage using Fluoro-Jade B histofluorescence and gliosis in the striatum after various durations of transient cerebral ischemia in gerbils. Neurochem. Res. 37, 826–834. Olavarria, J., Van Sluyters, R.C., Killackey, H.P., 1984. Evidence for the complementary organization of callosal and thalamic connections within rat somatosensory cortex. Brain Res. 291, 364–368. Petito, C.K., Feldmann, E., Pulsinelli, W.A., Plum, F., 1987. Delayed hippocampal damage in humans following cardiorespiratory arrest. Neurology 37, 1281–1286. Petito, C.K., Morgello, S., Felix, J.C., Lesser, M.L., 1990. The two patterns of reactive astrocytosis in postischemic rat brain. J. Cereb. Blood Flow Metab. 10, 850–859. Rorke, L.B., 1992. Anatomical features of the developing brain implicated in pathogenesis of hypoxic-ischemic injury. Brain Pathol. 2, 211–221. Schmidt-Kastner, R., Freund, T.F., 1991. Selective vulnerability of the hippocampus in brain ischemia. Neuroscience 40, 599–636. Schmued, L.C., Hopkins, K.J., 2000. Fluoro-Jade B: a high affinity fluorescent marker for the localization of neuronal degeneration. Brain Res. 874, 123–130. Schwartz, M., Butovsky, O., Bruck, W., Hanisch, U.K., 2006. Microglial phenotype: is the commitment reversible?. Trends Neurosci. 29, 68–74. Sims, N.R., Zaidan, E., 1995. Biochemical changes associated with selective neuronal death following short-term cerebral ischaemia. Int. J. Biochem. Cell Biol. 27, 531–550. Suzuki, S., Tanaka, K., Nogawa, S., Nagata, E., Ito, D., Dembo, T., Fukuuchi, Y., 1999. Temporal profile and cellular localization of interleukin-6 protein after focal cerebral ischemia in rats. J. Cereb. Blood Flow Metab. 19, 1256–1262. Wu, C.W., van Gelderen, P., Hanakawa, T., Yaseen, Z., Cohen, L.G., 2005. Enduring representational plasticity after somatosensory stimulation. Neuroimage 27, 872–884. Yan, B.C., Choi, J.H., Yoo, K.Y., Lee, C.H., Hwang, I.K., You, S.G., Kang, I.J., Kim, J.D., Kim, D.J., Kim, Y.M., Won, M.H., 2011. Leptin’s neuroprotective action in experimental transient ischemic damage of the gerbil hippocampus is linked to altered leptin receptor immunoreactivity. J. Neurol. Sci. 303, 100–108. Yu, D.K., Yoo, K.Y., Shin, B.N., Kim, I.H., Park, J.H., Lee, C.H., Choi, J.H., Cho, Y.J., Kang, I.J., Kim, Y.M., Won, M.H., 2012. Neuronal damage in hippocampal subregions induced by various durations of transient cerebral ischemia in gerbils using Fluoro-Jade B histofluorescence. Brain Res. 1437, 50–57. Zhang, Y.B., Kan, M.Y., Yang, Z.H., Ding, W.L., Yi, J., Chen, H.Z., Lu, Y., 2009. Neuroprotective effects of N-stearoyltyrosine on transient global cerebral ischemia in gerbils. Brain Res. 1287, 146–156.